PMC:1540739 JSON TXT 9 Projects

Annnotations TAB TSV DIC JSON TextAE

Id	Subject	Object	Predicate	Lexical cue
T440	0-4	NN	denotes	RMCE
T442	0-92	sentence	denotes	RMCE-ASAP: a gene targeting method for ES and somatic cells to accelerate phenotype analyses
T443	4-5	HYPH	denotes	-
T441	5-9	NN	denotes	ASAP
T444	9-11	:	denotes	:
T445	11-12	DT	denotes	a
T447	13-17	NN	denotes	gene
T448	18-27	VBG	denotes	targeting
T446	28-34	NN	denotes	method
T449	35-38	IN	denotes	for
T450	39-41	NN	denotes	ES
T452	42-45	CC	denotes	and
T453	46-53	JJ	denotes	somatic
T451	54-59	NNS	denotes	cells
T454	60-62	TO	denotes	to
T455	63-73	VB	denotes	accelerate
T456	74-83	NN	denotes	phenotype
T457	84-92	NNS	denotes	analyses
T458	92-93	sentence	denotes
T460	103-105	IN	denotes	In
T459	103-280	sentence	denotes	In recent years, tremendous insight has been gained on p53 regulation by targeting mutations at the p53 locus using homologous recombination in ES cells to generate mutant mice.
T462	106-112	JJ	denotes	recent
T463	113-118	NNS	denotes	years
T464	118-120	,	denotes	,
T465	120-130	JJ	denotes	tremendous
T466	131-138	NN	denotes	insight
T467	139-142	VBZ	denotes	has
T468	143-147	VBN	denotes	been
T461	148-154	VBN	denotes	gained
T469	155-157	IN	denotes	on
T470	158-161	NN	denotes	p53
T471	162-172	NN	denotes	regulation
T472	173-175	IN	denotes	by
T473	176-185	VBG	denotes	targeting
T474	186-195	NNS	denotes	mutations
T475	196-198	IN	denotes	at
T476	199-202	DT	denotes	the
T478	203-206	NN	denotes	p53
T477	207-212	NN	denotes	locus
T479	213-218	VBG	denotes	using
T480	219-229	JJ	denotes	homologous
T481	230-243	NN	denotes	recombination
T482	244-246	IN	denotes	in
T483	247-249	NN	denotes	ES
T484	250-255	NNS	denotes	cells
T485	256-258	TO	denotes	to
T486	259-267	VB	denotes	generate
T487	268-274	JJ	denotes	mutant
T488	275-279	NNS	denotes	mice
T489	279-280	.	denotes	.
T490	280-352	sentence	denotes	Although informative, this approach is inefficient, slow and expensive.
T491	281-289	IN	denotes	Although
T492	290-301	JJ	denotes	informative
T494	301-303	,	denotes	,
T495	303-307	DT	denotes	this
T496	308-316	NN	denotes	approach
T493	317-319	VBZ	denotes	is
T497	320-331	JJ	denotes	inefficient
T498	331-333	,	denotes	,
T499	333-337	JJ	denotes	slow
T500	338-341	CC	denotes	and
T501	342-351	JJ	denotes	expensive
T502	351-352	.	denotes	.
T503	352-473	sentence	denotes	To facilitate targeting at the p53 locus, we developed an improved Recombinase-Mediated Cassette Exchange (RMCE) method.
T504	353-355	TO	denotes	To
T505	356-366	VB	denotes	facilitate
T507	367-376	NN	denotes	targeting
T508	377-379	IN	denotes	at
T509	380-383	DT	denotes	the
T511	384-387	NN	denotes	p53
T510	388-393	NN	denotes	locus
T512	393-395	,	denotes	,
T513	395-397	PRP	denotes	we
T506	398-407	VBD	denotes	developed
T514	408-410	DT	denotes	an
T516	411-419	VBN	denotes	improved
T517	420-431	NN	denotes	Recombinase
T519	431-432	HYPH	denotes	-
T518	432-440	JJ	denotes	Mediated
T521	441-449	NNP	denotes	Cassette
T520	450-458	NNP	denotes	Exchange
T522	459-460	-LRB-	denotes	(
T523	460-464	NN	denotes	RMCE
T524	464-465	-RRB-	denotes	)
T515	466-472	NN	denotes	method
T525	472-473	.	denotes	.
T526	473-571	sentence	denotes	Our approach enables efficient targeting in ES cells to facilitate the production of mutant mice.
T527	474-477	PRP$	denotes	Our
T528	478-486	NN	denotes	approach
T529	487-494	VBZ	denotes	enables
T530	495-504	JJ	denotes	efficient
T531	505-514	NN	denotes	targeting
T532	515-517	IN	denotes	in
T533	518-520	NN	denotes	ES
T534	521-526	NNS	denotes	cells
T535	527-529	TO	denotes	to
T536	530-540	VB	denotes	facilitate
T537	541-544	DT	denotes	the
T538	545-555	NN	denotes	production
T539	556-558	IN	denotes	of
T540	559-565	JJ	denotes	mutant
T541	566-570	NNS	denotes	mice
T542	570-571	.	denotes	.
T543	571-688	sentence	denotes	But more importantly, the approach was Adapted for targeting in Somatic cells to Accelerate Phenotyping (RMCE-ASAP).
T544	572-575	CC	denotes	But
T546	576-580	RBR	denotes	more
T547	581-592	RB	denotes	importantly
T548	592-594	,	denotes	,
T549	594-597	DT	denotes	the
T550	598-606	NN	denotes	approach
T551	607-610	VBD	denotes	was
T545	611-618	VBN	denotes	Adapted
T552	619-622	IN	denotes	for
T553	623-632	NN	denotes	targeting
T554	633-635	IN	denotes	in
T555	636-643	JJ	denotes	Somatic
T556	644-649	NNS	denotes	cells
T557	650-652	TO	denotes	to
T558	653-663	VB	denotes	Accelerate
T559	664-675	NN	denotes	Phenotyping
T560	676-677	-LRB-	denotes	(
T561	677-681	NN	denotes	RMCE
T563	681-682	HYPH	denotes	-
T562	682-686	NN	denotes	ASAP
T564	686-687	-RRB-	denotes	)
T565	687-688	.	denotes	.
T566	688-869	sentence	denotes	We provide proof-of-concept for this at the p53 locus, by showing efficient targeting in fibroblasts, and rapid phenotypic read-out of a recessive mutation after a single exchange.
T567	689-691	PRP	denotes	We
T568	692-699	VBP	denotes	provide
T569	700-705	NN	denotes	proof
T570	705-706	HYPH	denotes	-
T571	706-708	IN	denotes	of
T572	708-709	HYPH	denotes	-
T573	709-716	NN	denotes	concept
T574	717-720	IN	denotes	for
T575	721-725	DT	denotes	this
T576	726-728	IN	denotes	at
T577	729-732	DT	denotes	the
T579	733-736	NN	denotes	p53
T578	737-742	NN	denotes	locus
T580	742-744	,	denotes	,
T581	744-746	IN	denotes	by
T582	747-754	VBG	denotes	showing
T583	755-764	JJ	denotes	efficient
T584	765-774	NN	denotes	targeting
T585	775-777	IN	denotes	in
T586	778-789	NNS	denotes	fibroblasts
T587	789-791	,	denotes	,
T588	791-794	CC	denotes	and
T589	795-800	JJ	denotes	rapid
T591	801-811	JJ	denotes	phenotypic
T592	812-816	NN	denotes	read
T593	816-817	HYPH	denotes	-
T590	817-820	NN	denotes	out
T594	821-823	IN	denotes	of
T595	824-825	DT	denotes	a
T597	826-835	JJ	denotes	recessive
T596	836-844	NN	denotes	mutation
T598	845-850	IN	denotes	after
T599	851-852	DT	denotes	a
T601	853-859	JJ	denotes	single
T600	860-868	NN	denotes	exchange
T602	868-869	.	denotes	.
T603	869-1056	sentence	denotes	RMCE-ASAP combines inverted heterologous recombinase target sites, a positive/negative selection marker that preserves the germline capacity of ES cells, and the power of mouse genetics.
T604	870-874	NN	denotes	RMCE
T606	874-875	HYPH	denotes	-
T605	875-879	NN	denotes	ASAP
T607	880-888	VBZ	denotes	combines
T608	889-897	JJ	denotes	inverted
T610	898-910	JJ	denotes	heterologous
T611	911-922	NN	denotes	recombinase
T612	923-929	NN	denotes	target
T609	930-935	NNS	denotes	sites
T613	935-937	,	denotes	,
T614	937-938	DT	denotes	a
T616	939-947	JJ	denotes	positive
T618	947-948	HYPH	denotes	/
T617	948-956	JJ	denotes	negative
T619	957-966	NN	denotes	selection
T615	967-973	NN	denotes	marker
T620	974-978	WDT	denotes	that
T621	979-988	VBZ	denotes	preserves
T622	989-992	DT	denotes	the
T624	993-1001	NN	denotes	germline
T623	1002-1010	NN	denotes	capacity
T625	1011-1013	IN	denotes	of
T626	1014-1016	NN	denotes	ES
T627	1017-1022	NNS	denotes	cells
T628	1022-1024	,	denotes	,
T629	1024-1027	CC	denotes	and
T630	1028-1031	DT	denotes	the
T631	1032-1037	NN	denotes	power
T632	1038-1040	IN	denotes	of
T633	1041-1046	NN	denotes	mouse
T634	1047-1055	NN	denotes	genetics
T635	1055-1056	.	denotes	.
T636	1056-1128	sentence	denotes	These general principles should make RMCE-ASAP applicable to any locus.
T637	1057-1062	DT	denotes	These
T639	1063-1070	JJ	denotes	general
T638	1071-1081	NNS	denotes	principles
T641	1082-1088	MD	denotes	should
T640	1089-1093	VB	denotes	make
T642	1094-1098	NN	denotes	RMCE
T644	1098-1099	HYPH	denotes	-
T643	1099-1103	NN	denotes	ASAP
T645	1104-1114	JJ	denotes	applicable
T646	1115-1117	IN	denotes	to
T647	1118-1121	DT	denotes	any
T648	1122-1127	NN	denotes	locus
T649	1127-1128	.	denotes	.
T2571	1143-1146	NN	denotes	p53
T2572	1147-1149	VBZ	denotes	is
T2573	1150-1153	CD	denotes	one
T2574	1154-1156	IN	denotes	of
T2575	1157-1160	DT	denotes	the
T2577	1161-1165	RBS	denotes	most
T2578	1166-1172	RB	denotes	highly
T2579	1173-1181	JJ	denotes	analyzed
T2576	1182-1190	NN	denotes	proteins
T2580	1191-1194	IN	denotes	for
T2581	1195-1198	DT	denotes	the
T2583	1199-1203	JJ	denotes	past
T2584	1204-1206	CD	denotes	25
T2582	1207-1212	NNS	denotes	years
T2585	1212-1213	.	denotes	.
T2586	1213-1375	sentence	denotes	Studies in cultured cells, often relying on the transfection of plasmids expressing various p53 mutants, have established models to explain how p53 is regulated.
T2587	1214-1221	NNS	denotes	Studies
T2589	1222-1224	IN	denotes	in
T2590	1225-1233	VBN	denotes	cultured
T2591	1234-1239	NNS	denotes	cells
T2592	1239-1241	,	denotes	,
T2593	1241-1246	RB	denotes	often
T2594	1247-1254	VBG	denotes	relying
T2595	1255-1257	IN	denotes	on
T2596	1258-1261	DT	denotes	the
T2597	1262-1274	NN	denotes	transfection
T2598	1275-1277	IN	denotes	of
T2599	1278-1286	NNS	denotes	plasmids
T2600	1287-1297	VBG	denotes	expressing
T2601	1298-1305	JJ	denotes	various
T2603	1306-1309	NN	denotes	p53
T2602	1310-1317	NNS	denotes	mutants
T2604	1317-1319	,	denotes	,
T2605	1319-1323	VBD	denotes	have
T2588	1324-1335	VBN	denotes	established
T2606	1336-1342	NNS	denotes	models
T2607	1343-1345	TO	denotes	to
T2608	1346-1353	VB	denotes	explain
T2609	1354-1357	WRB	denotes	how
T2611	1358-1361	NN	denotes	p53
T2612	1362-1364	VBZ	denotes	is
T2610	1365-1374	VBN	denotes	regulated
T2613	1374-1375	.	denotes	.
T2614	1375-1565	sentence	denotes	In recent years, some of these models were tested in vivo by targeting subtle mutations at the p53 locus using homologous recombination in embryonic stem (ES) cells to generate mutant mice.
T2615	1376-1378	IN	denotes	In
T2617	1379-1385	JJ	denotes	recent
T2618	1386-1391	NNS	denotes	years
T2619	1391-1393	,	denotes	,
T2620	1393-1397	DT	denotes	some
T2621	1398-1400	IN	denotes	of
T2622	1401-1406	DT	denotes	these
T2623	1407-1413	NNS	denotes	models
T2624	1414-1418	VBD	denotes	were
T2616	1419-1425	VBN	denotes	tested
T2625	1426-1428	FW	denotes	in
T2626	1429-1433	FW	denotes	vivo
T2627	1434-1436	IN	denotes	by
T2628	1437-1446	VBG	denotes	targeting
T2629	1447-1453	JJ	denotes	subtle
T2630	1454-1463	NNS	denotes	mutations
T2631	1464-1466	IN	denotes	at
T2632	1467-1470	DT	denotes	the
T2634	1471-1474	NN	denotes	p53
T2633	1475-1480	NN	denotes	locus
T2635	1481-1486	VBG	denotes	using
T2636	1487-1497	JJ	denotes	homologous
T2637	1498-1511	NN	denotes	recombination
T2638	1512-1514	IN	denotes	in
T2639	1515-1524	JJ	denotes	embryonic
T2640	1525-1529	NN	denotes	stem
T2642	1530-1531	-LRB-	denotes	(
T2643	1531-1533	NN	denotes	ES
T2644	1533-1534	-RRB-	denotes	)
T2641	1535-1540	NNS	denotes	cells
T2645	1541-1543	TO	denotes	to
T2646	1544-1552	VB	denotes	generate
T2647	1553-1559	JJ	denotes	mutant
T2648	1560-1564	NNS	denotes	mice
T2649	1564-1565	.	denotes	.
T2650	1565-1768	sentence	denotes	The strength of this approach is that mutations are tested in a genomic setting and expressed from the endogenous promoter, ensuring physiological expression levels and correct spatio-temporal profiles.
T2651	1566-1569	DT	denotes	The
T2652	1570-1578	NN	denotes	strength
T2654	1579-1581	IN	denotes	of
T2655	1582-1586	DT	denotes	this
T2656	1587-1595	NN	denotes	approach
T2653	1596-1598	VBZ	denotes	is
T2657	1599-1603	IN	denotes	that
T2659	1604-1613	NNS	denotes	mutations
T2660	1614-1617	VBP	denotes	are
T2658	1618-1624	VBN	denotes	tested
T2661	1625-1627	IN	denotes	in
T2662	1628-1629	DT	denotes	a
T2664	1630-1637	JJ	denotes	genomic
T2663	1638-1645	NN	denotes	setting
T2665	1646-1649	CC	denotes	and
T2666	1650-1659	VBN	denotes	expressed
T2667	1660-1664	IN	denotes	from
T2668	1665-1668	DT	denotes	the
T2670	1669-1679	JJ	denotes	endogenous
T2669	1680-1688	NN	denotes	promoter
T2671	1688-1690	,	denotes	,
T2672	1690-1698	VBG	denotes	ensuring
T2673	1699-1712	JJ	denotes	physiological
T2675	1713-1723	NN	denotes	expression
T2674	1724-1730	NNS	denotes	levels
T2676	1731-1734	CC	denotes	and
T2677	1735-1742	JJ	denotes	correct
T2679	1743-1749	JJ	denotes	spatio
T2681	1749-1750	HYPH	denotes	-
T2680	1750-1758	JJ	denotes	temporal
T2678	1759-1767	NNS	denotes	profiles
T2682	1767-1768	.	denotes	.
T2683	1768-2034	sentence	denotes	As significant differences between phenotypes from targeted p53 mutants in vivo and transfection data were observed [e.g. Refs (1–5)], more targeted mutations need to be generated and analyzed in multiple tissues to formulate more accurate models of p53 regulation.
T2684	1769-1771	IN	denotes	As
T2686	1772-1783	JJ	denotes	significant
T2687	1784-1795	NNS	denotes	differences
T2688	1796-1803	IN	denotes	between
T2689	1804-1814	NNS	denotes	phenotypes
T2690	1815-1819	IN	denotes	from
T2691	1820-1828	VBN	denotes	targeted
T2693	1829-1832	NN	denotes	p53
T2692	1833-1840	NNS	denotes	mutants
T2694	1841-1843	FW	denotes	in
T2695	1844-1848	FW	denotes	vivo
T2696	1849-1852	CC	denotes	and
T2697	1853-1865	NN	denotes	transfection
T2698	1866-1870	NNS	denotes	data
T2699	1871-1875	VBD	denotes	were
T2685	1876-1884	VBN	denotes	observed
T2701	1885-1886	-LRB-	denotes	[
T2703	1886-1890	FW	denotes	e.g.
T2704	1891-1895	NNS	denotes	Refs
T2705	1896-1897	-LRB-	denotes	(
T2702	1897-1898	CD	denotes	1
T2706	1898-1899	SYM	denotes	–
T2707	1899-1900	CD	denotes	5
T2708	1900-1901	-RRB-	denotes	)
T2709	1901-1902	-RRB-	denotes	]
T2710	1902-1904	,	denotes	,
T2711	1904-1908	RBR	denotes	more
T2712	1909-1917	VBN	denotes	targeted
T2713	1918-1927	NNS	denotes	mutations
T2700	1928-1932	VBP	denotes	need
T2714	1933-1935	TO	denotes	to
T2716	1936-1938	VB	denotes	be
T2715	1939-1948	VBN	denotes	generated
T2717	1949-1952	CC	denotes	and
T2718	1953-1961	VBN	denotes	analyzed
T2719	1962-1964	IN	denotes	in
T2720	1965-1973	JJ	denotes	multiple
T2721	1974-1981	NNS	denotes	tissues
T2722	1982-1984	TO	denotes	to
T2723	1985-1994	VB	denotes	formulate
T2724	1995-1999	RBR	denotes	more
T2725	2000-2008	JJ	denotes	accurate
T2726	2009-2015	NNS	denotes	models
T2727	2016-2018	IN	denotes	of
T2728	2019-2022	NN	denotes	p53
T2729	2023-2033	NN	denotes	regulation
T2730	2033-2034	.	denotes	.
T2731	2034-2555	sentence	denotes	However, using homologous recombination in ES cells to generate mutant mice is an inefficient, slow and expensive method because (i) homologous recombination typically occurs at low frequency in ES cells, requiring sophisticated selection schemes and screening of hundreds of clones to identify the desired mutant; (ii) large (15–20 kb) plasmids, often difficult to clone, are required to increase targeting efficiency and (iii) breeding mice to homozygosity and housing a mouse colony generate further delays and costs.
T2732	2035-2042	RB	denotes	However
T2734	2042-2044	,	denotes	,
T2735	2044-2049	VBG	denotes	using
T2736	2050-2060	JJ	denotes	homologous
T2737	2061-2074	NN	denotes	recombination
T2738	2075-2077	IN	denotes	in
T2739	2078-2080	NN	denotes	ES
T2740	2081-2086	NNS	denotes	cells
T2741	2087-2089	TO	denotes	to
T2742	2090-2098	VB	denotes	generate
T2743	2099-2105	JJ	denotes	mutant
T2744	2106-2110	NNS	denotes	mice
T2733	2111-2113	VBZ	denotes	is
T2745	2114-2116	DT	denotes	an
T2747	2117-2128	JJ	denotes	inefficient
T2748	2128-2130	,	denotes	,
T2749	2130-2134	JJ	denotes	slow
T2750	2135-2138	CC	denotes	and
T2751	2139-2148	JJ	denotes	expensive
T2746	2149-2155	NN	denotes	method
T2752	2156-2163	IN	denotes	because
T2754	2164-2165	-LRB-	denotes	(
T2755	2165-2166	LS	denotes	i
T2756	2166-2167	-RRB-	denotes	)
T2757	2168-2178	JJ	denotes	homologous
T2758	2179-2192	NN	denotes	recombination
T2759	2193-2202	RB	denotes	typically
T2753	2203-2209	VBZ	denotes	occurs
T2760	2210-2212	IN	denotes	at
T2761	2213-2216	JJ	denotes	low
T2762	2217-2226	NN	denotes	frequency
T2763	2227-2229	IN	denotes	in
T2764	2230-2232	NN	denotes	ES
T2765	2233-2238	NNS	denotes	cells
T2766	2238-2240	,	denotes	,
T2767	2240-2249	VBG	denotes	requiring
T2768	2250-2263	JJ	denotes	sophisticated
T2770	2264-2273	NN	denotes	selection
T2769	2274-2281	NNS	denotes	schemes
T2771	2282-2285	CC	denotes	and
T2772	2286-2295	NN	denotes	screening
T2773	2296-2298	IN	denotes	of
T2774	2299-2307	NNS	denotes	hundreds
T2775	2308-2310	IN	denotes	of
T2776	2311-2317	NNS	denotes	clones
T2777	2318-2320	TO	denotes	to
T2778	2321-2329	VB	denotes	identify
T2779	2330-2333	DT	denotes	the
T2781	2334-2341	JJ	denotes	desired
T2780	2342-2348	NN	denotes	mutant
T2782	2348-2349	:	denotes	;
T2783	2350-2351	-LRB-	denotes	(
T2784	2351-2353	LS	denotes	ii
T2786	2353-2354	-RRB-	denotes	)
T2787	2355-2360	JJ	denotes	large
T2788	2361-2362	-LRB-	denotes	(
T2790	2362-2364	CD	denotes	15
T2792	2364-2365	SYM	denotes	–
T2791	2365-2367	CD	denotes	20
T2789	2368-2370	NN	denotes	kb
T2793	2370-2371	-RRB-	denotes	)
T2785	2372-2380	NNS	denotes	plasmids
T2795	2380-2382	,	denotes	,
T2796	2382-2387	RB	denotes	often
T2797	2388-2397	JJ	denotes	difficult
T2798	2398-2400	TO	denotes	to
T2799	2401-2406	VB	denotes	clone
T2800	2406-2408	,	denotes	,
T2801	2408-2411	VBP	denotes	are
T2794	2412-2420	VBN	denotes	required
T2802	2421-2423	TO	denotes	to
T2803	2424-2432	VB	denotes	increase
T2804	2433-2442	VBG	denotes	targeting
T2805	2443-2453	NN	denotes	efficiency
T2806	2454-2457	CC	denotes	and
T2807	2458-2459	-LRB-	denotes	(
T2808	2459-2462	LS	denotes	iii
T2810	2462-2463	-RRB-	denotes	)
T2811	2464-2472	VBG	denotes	breeding
T2812	2473-2477	NNS	denotes	mice
T2813	2478-2480	IN	denotes	to
T2814	2481-2493	NN	denotes	homozygosity
T2815	2494-2497	CC	denotes	and
T2816	2498-2505	VBG	denotes	housing
T2817	2506-2507	DT	denotes	a
T2819	2508-2513	NN	denotes	mouse
T2818	2514-2520	NN	denotes	colony
T2809	2521-2529	VBP	denotes	generate
T2820	2530-2537	JJ	denotes	further
T2821	2538-2544	NNS	denotes	delays
T2822	2545-2548	CC	denotes	and
T2823	2549-2554	NNS	denotes	costs
T2824	2554-2555	.	denotes	.
T2825	2555-2669	sentence	denotes	Such limitations make the repeated targeting of a locus a technically daunting and economically impractical task.
T2826	2556-2560	JJ	denotes	Such
T2827	2561-2572	NNS	denotes	limitations
T2828	2573-2577	VBP	denotes	make
T2829	2578-2581	DT	denotes	the
T2831	2582-2590	VBN	denotes	repeated
T2830	2591-2600	NN	denotes	targeting
T2833	2601-2603	IN	denotes	of
T2834	2604-2605	DT	denotes	a
T2835	2606-2611	NN	denotes	locus
T2836	2612-2613	DT	denotes	a
T2837	2614-2625	RB	denotes	technically
T2838	2626-2634	VBG	denotes	daunting
T2839	2635-2638	CC	denotes	and
T2840	2639-2651	RB	denotes	economically
T2841	2652-2663	JJ	denotes	impractical
T2832	2664-2668	NN	denotes	task
T2842	2668-2669	.	denotes	.
T2843	2669-2782	sentence	denotes	Improvements in current technologies are needed to enable such analyses to be applied to the p53 or other genes.
T2844	2670-2682	NNS	denotes	Improvements
T2846	2683-2685	IN	denotes	in
T2847	2686-2693	JJ	denotes	current
T2848	2694-2706	NNS	denotes	technologies
T2849	2707-2710	VBP	denotes	are
T2845	2711-2717	VBN	denotes	needed
T2850	2718-2720	TO	denotes	to
T2851	2721-2727	VB	denotes	enable
T2852	2728-2732	JJ	denotes	such
T2853	2733-2741	NNS	denotes	analyses
T2854	2742-2744	TO	denotes	to
T2856	2745-2747	VB	denotes	be
T2855	2748-2755	VBN	denotes	applied
T2857	2756-2758	IN	denotes	to
T2858	2759-2762	DT	denotes	the
T2859	2763-2766	NN	denotes	p53
T2860	2767-2769	CC	denotes	or
T2861	2770-2775	JJ	denotes	other
T2862	2776-2781	NNS	denotes	genes
T2863	2781-2782	.	denotes	.
T2864	2782-2876	sentence	denotes	Developing methods to increase targeting efficiency in ES cells is clearly an important goal.
T2865	2783-2793	VBG	denotes	Developing
T2867	2794-2801	NNS	denotes	methods
T2868	2802-2804	TO	denotes	to
T2869	2805-2813	VB	denotes	increase
T2870	2814-2823	VBG	denotes	targeting
T2871	2824-2834	NN	denotes	efficiency
T2872	2835-2837	IN	denotes	in
T2873	2838-2840	NN	denotes	ES
T2874	2841-2846	NNS	denotes	cells
T2866	2847-2849	VBZ	denotes	is
T2875	2850-2857	RB	denotes	clearly
T2876	2858-2860	DT	denotes	an
T2878	2861-2870	JJ	denotes	important
T2877	2871-2875	NN	denotes	goal
T2879	2875-2876	.	denotes	.
T2880	2876-2977	sentence	denotes	In addition, efficient methods for gene targeting in fibroblasts could expedite phenotypic analyses.
T2881	2877-2879	IN	denotes	In
T2883	2880-2888	NN	denotes	addition
T2884	2888-2890	,	denotes	,
T2885	2890-2899	JJ	denotes	efficient
T2886	2900-2907	NNS	denotes	methods
T2887	2908-2911	IN	denotes	for
T2888	2912-2916	NN	denotes	gene
T2889	2917-2926	NN	denotes	targeting
T2890	2927-2929	IN	denotes	in
T2891	2930-2941	NNS	denotes	fibroblasts
T2892	2942-2947	MD	denotes	could
T2882	2948-2956	VB	denotes	expedite
T2893	2957-2967	JJ	denotes	phenotypic
T2894	2968-2976	NNS	denotes	analyses
T2895	2976-2977	.	denotes	.
T2896	2977-3087	sentence	denotes	Indeed, siRNAs in fibroblasts often provide a faster read-out than equivalent gene knock-outs in animals (6).
T2897	2978-2984	RB	denotes	Indeed
T2899	2984-2986	,	denotes	,
T2900	2986-2992	NNS	denotes	siRNAs
T2901	2993-2995	IN	denotes	in
T2902	2996-3007	NNS	denotes	fibroblasts
T2903	3008-3013	RB	denotes	often
T2898	3014-3021	VBP	denotes	provide
T2904	3022-3023	DT	denotes	a
T2906	3024-3030	JJR	denotes	faster
T2907	3031-3035	NN	denotes	read
T2908	3035-3036	HYPH	denotes	-
T2905	3036-3039	NN	denotes	out
T2909	3040-3044	IN	denotes	than
T2910	3045-3055	JJ	denotes	equivalent
T2912	3056-3060	NN	denotes	gene
T2913	3061-3066	NN	denotes	knock
T2914	3066-3067	HYPH	denotes	-
T2911	3067-3071	NNS	denotes	outs
T2915	3072-3074	IN	denotes	in
T2916	3075-3082	NNS	denotes	animals
T2917	3083-3084	-LRB-	denotes	(
T2918	3084-3085	CD	denotes	6
T2919	3085-3086	-RRB-	denotes	)
T2920	3086-3087	.	denotes	.
T2921	3087-3223	sentence	denotes	However, modeling most disease-associated mutations requires generating subtle mutations, not knock-outs or reduced expression alleles.
T2922	3088-3095	RB	denotes	However
T2924	3095-3097	,	denotes	,
T2925	3097-3105	VBG	denotes	modeling
T2926	3106-3110	RBS	denotes	most
T2928	3111-3118	NN	denotes	disease
T2930	3118-3119	HYPH	denotes	-
T2929	3119-3129	JJ	denotes	associated
T2927	3130-3139	NNS	denotes	mutations
T2923	3140-3148	VBZ	denotes	requires
T2931	3149-3159	VBG	denotes	generating
T2932	3160-3166	JJ	denotes	subtle
T2933	3167-3176	NNS	denotes	mutations
T2934	3176-3178	,	denotes	,
T2935	3178-3181	CC	denotes	not
T2936	3182-3187	NN	denotes	knock
T2938	3187-3188	HYPH	denotes	-
T2937	3188-3192	NNS	denotes	outs
T2939	3193-3195	CC	denotes	or
T2940	3196-3203	VBN	denotes	reduced
T2941	3204-3214	NN	denotes	expression
T2942	3215-3222	NNS	denotes	alleles
T2943	3222-3223	.	denotes	.
T2944	3223-3415	sentence	denotes	Targeting point mutations in fibroblasts by homologous recombination is extremely inefficient, and targeting both alleles is required to reveal the phenotype of recessive autosomal mutations.
T2945	3224-3233	VBG	denotes	Targeting
T2947	3234-3239	NN	denotes	point
T2948	3240-3249	NNS	denotes	mutations
T2949	3250-3252	IN	denotes	in
T2950	3253-3264	NNS	denotes	fibroblasts
T2951	3265-3267	IN	denotes	by
T2952	3268-3278	JJ	denotes	homologous
T2953	3279-3292	NN	denotes	recombination
T2946	3293-3295	VBZ	denotes	is
T2954	3296-3305	RB	denotes	extremely
T2955	3306-3317	JJ	denotes	inefficient
T2956	3317-3319	,	denotes	,
T2957	3319-3322	CC	denotes	and
T2958	3323-3332	VBG	denotes	targeting
T2960	3333-3337	DT	denotes	both
T2961	3338-3345	NNS	denotes	alleles
T2962	3346-3348	VBZ	denotes	is
T2959	3349-3357	VBN	denotes	required
T2963	3358-3360	TO	denotes	to
T2964	3361-3367	VB	denotes	reveal
T2965	3368-3371	DT	denotes	the
T2966	3372-3381	NN	denotes	phenotype
T2967	3382-3384	IN	denotes	of
T2968	3385-3394	JJ	denotes	recessive
T2969	3395-3404	JJ	denotes	autosomal
T2970	3405-3414	NNS	denotes	mutations
T2971	3414-3415	.	denotes	.
T2972	3415-3533	sentence	denotes	Here we report an approach that enables highly efficient targeting at the p53 locus in both ES cells and fibroblasts.
T2973	3416-3420	RB	denotes	Here
T2975	3421-3423	PRP	denotes	we
T2974	3424-3430	VBP	denotes	report
T2976	3431-3433	DT	denotes	an
T2977	3434-3442	NN	denotes	approach
T2978	3443-3447	WDT	denotes	that
T2979	3448-3455	VBZ	denotes	enables
T2980	3456-3462	RB	denotes	highly
T2981	3463-3472	JJ	denotes	efficient
T2982	3473-3482	NN	denotes	targeting
T2983	3483-3485	IN	denotes	at
T2984	3486-3489	DT	denotes	the
T2986	3490-3493	NN	denotes	p53
T2985	3494-3499	NN	denotes	locus
T2987	3500-3502	IN	denotes	in
T2988	3503-3507	CC	denotes	both
T2990	3508-3510	NN	denotes	ES
T2989	3511-3516	NNS	denotes	cells
T2991	3517-3520	CC	denotes	and
T2992	3521-3532	NNS	denotes	fibroblasts
T2993	3532-3533	.	denotes	.
T2994	3533-3829	sentence	denotes	Recombinase-Mediated Cassette Exchange (RMCE) approaches were developed to improve targeting efficiency using a two-step process: the gene of interest is first replaced by a selection cassette flanked by recombinase target sites (e.g. loxP sites for Cre recombinase, to create a ‘floxed’ locus).
T2995	3534-3545	NN	denotes	Recombinase
T2997	3545-3546	HYPH	denotes	-
T2996	3546-3554	JJ	denotes	Mediated
T2999	3555-3563	NNP	denotes	Cassette
T2998	3564-3572	NNP	denotes	Exchange
T3001	3573-3574	-LRB-	denotes	(
T3002	3574-3578	NN	denotes	RMCE
T3003	3578-3579	-RRB-	denotes	)
T3000	3580-3590	NNS	denotes	approaches
T3005	3591-3595	VBD	denotes	were
T3004	3596-3605	VBN	denotes	developed
T3007	3606-3608	TO	denotes	to
T3008	3609-3616	VB	denotes	improve
T3009	3617-3626	VBG	denotes	targeting
T3010	3627-3637	NN	denotes	efficiency
T3011	3638-3643	VBG	denotes	using
T3012	3644-3645	DT	denotes	a
T3014	3646-3649	CD	denotes	two
T3016	3649-3650	HYPH	denotes	-
T3015	3650-3654	NN	denotes	step
T3013	3655-3662	NN	denotes	process
T3017	3662-3664	:	denotes	:
T3018	3664-3667	DT	denotes	the
T3019	3668-3672	NN	denotes	gene
T3020	3673-3675	IN	denotes	of
T3021	3676-3684	NN	denotes	interest
T3022	3685-3687	VBZ	denotes	is
T3023	3688-3693	RB	denotes	first
T3006	3694-3702	VBN	denotes	replaced
T3024	3703-3705	IN	denotes	by
T3025	3706-3707	DT	denotes	a
T3027	3708-3717	NN	denotes	selection
T3026	3718-3726	NN	denotes	cassette
T3028	3727-3734	VBN	denotes	flanked
T3029	3735-3737	IN	denotes	by
T3030	3738-3749	NN	denotes	recombinase
T3032	3750-3756	NN	denotes	target
T3031	3757-3762	NNS	denotes	sites
T3033	3763-3764	-LRB-	denotes	(
T3035	3764-3768	FW	denotes	e.g.
T3036	3769-3773	NN	denotes	loxP
T3034	3774-3779	NNS	denotes	sites
T3037	3780-3783	IN	denotes	for
T3038	3784-3787	NN	denotes	Cre
T3039	3788-3799	NN	denotes	recombinase
T3040	3799-3801	,	denotes	,
T3041	3801-3803	TO	denotes	to
T3042	3804-3810	VB	denotes	create
T3043	3811-3812	DT	denotes	a
T3045	3813-3814	``	denotes	‘
T3046	3814-3820	JJ	denotes	floxed
T3047	3820-3821	''	denotes	’
T3044	3822-3827	NN	denotes	locus
T3048	3827-3828	-RRB-	denotes	)
T3049	3828-3829	.	denotes	.
T3050	3829-3989	sentence	denotes	Then, Cre-mediated recombination in the presence of a cassette containing a floxed mutant allele removes the resident sequence and inserts the mutant gene (7).
T3051	3830-3834	RB	denotes	Then
T3053	3834-3836	,	denotes	,
T3054	3836-3839	NN	denotes	Cre
T3056	3839-3840	HYPH	denotes	-
T3055	3840-3848	JJ	denotes	mediated
T3057	3849-3862	NN	denotes	recombination
T3058	3863-3865	IN	denotes	in
T3059	3866-3869	DT	denotes	the
T3060	3870-3878	NN	denotes	presence
T3061	3879-3881	IN	denotes	of
T3062	3882-3883	DT	denotes	a
T3063	3884-3892	NN	denotes	cassette
T3064	3893-3903	VBG	denotes	containing
T3065	3904-3905	DT	denotes	a
T3067	3906-3912	VBN	denotes	floxed
T3068	3913-3919	JJ	denotes	mutant
T3066	3920-3926	NN	denotes	allele
T3052	3927-3934	VBZ	denotes	removes
T3069	3935-3938	DT	denotes	the
T3071	3939-3947	JJ	denotes	resident
T3070	3948-3956	NN	denotes	sequence
T3072	3957-3960	CC	denotes	and
T3073	3961-3968	VBZ	denotes	inserts
T3074	3969-3972	DT	denotes	the
T3076	3973-3979	JJ	denotes	mutant
T3075	3980-3984	NN	denotes	gene
T3077	3985-3986	-LRB-	denotes	(
T3078	3986-3987	CD	denotes	7
T3079	3987-3988	-RRB-	denotes	)
T3080	3988-3989	.	denotes	.
T3081	3989-4098	sentence	denotes	Previously, technical difficulties have prevented RMCE from being applied routinely to generate mutant mice.
T3082	3990-4000	RB	denotes	Previously
T3084	4000-4002	,	denotes	,
T3085	4002-4011	JJ	denotes	technical
T3086	4012-4024	NNS	denotes	difficulties
T3087	4025-4029	VBD	denotes	have
T3083	4030-4039	VBN	denotes	prevented
T3088	4040-4044	NN	denotes	RMCE
T3089	4045-4049	IN	denotes	from
T3090	4050-4055	VBG	denotes	being
T3091	4056-4063	VBN	denotes	applied
T3092	4064-4073	RB	denotes	routinely
T3093	4074-4076	TO	denotes	to
T3094	4077-4085	VB	denotes	generate
T3095	4086-4092	JJ	denotes	mutant
T3096	4093-4097	NNS	denotes	mice
T3097	4097-4098	.	denotes	.
T3098	4098-4244	sentence	denotes	For example, exchanges using cassettes with directly repeated loxP sites were inefficient because excisions dominated the intended exchanges (8).
T3099	4099-4102	IN	denotes	For
T3101	4103-4110	NN	denotes	example
T3102	4110-4112	,	denotes	,
T3103	4112-4121	NNS	denotes	exchanges
T3104	4122-4127	VBG	denotes	using
T3105	4128-4137	NNS	denotes	cassettes
T3106	4138-4142	IN	denotes	with
T3107	4143-4151	RB	denotes	directly
T3108	4152-4160	VBN	denotes	repeated
T3110	4161-4165	NN	denotes	loxP
T3109	4166-4171	NNS	denotes	sites
T3100	4172-4176	VBD	denotes	were
T3111	4177-4188	JJ	denotes	inefficient
T3112	4189-4196	IN	denotes	because
T3114	4197-4206	NNS	denotes	excisions
T3113	4207-4216	VBD	denotes	dominated
T3115	4217-4220	DT	denotes	the
T3117	4221-4229	VBN	denotes	intended
T3116	4230-4239	NNS	denotes	exchanges
T3118	4240-4241	-LRB-	denotes	(
T3119	4241-4242	CD	denotes	8
T3120	4242-4243	-RRB-	denotes	)
T3121	4243-4244	.	denotes	.
T3122	4244-4512	sentence	denotes	loxP sites with different sequences were generated to overcome this problem, but these sites also underwent intramolecular recombination, making RMCE efficient only if the replacement cassette contained a marker enabling selection of the desired recombinant (7,9–12).
T3123	4245-4249	NN	denotes	loxP
T3124	4250-4255	NNS	denotes	sites
T3126	4256-4260	IN	denotes	with
T3127	4261-4270	JJ	denotes	different
T3128	4271-4280	NNS	denotes	sequences
T3129	4281-4285	VBD	denotes	were
T3125	4286-4295	VBN	denotes	generated
T3130	4296-4298	TO	denotes	to
T3131	4299-4307	VB	denotes	overcome
T3132	4308-4312	DT	denotes	this
T3133	4313-4320	NN	denotes	problem
T3134	4320-4322	,	denotes	,
T3135	4322-4325	CC	denotes	but
T3136	4326-4331	DT	denotes	these
T3137	4332-4337	NNS	denotes	sites
T3139	4338-4342	RB	denotes	also
T3138	4343-4352	VBD	denotes	underwent
T3140	4353-4367	JJ	denotes	intramolecular
T3141	4368-4381	NN	denotes	recombination
T3142	4381-4383	,	denotes	,
T3143	4383-4389	VBG	denotes	making
T3144	4390-4394	NN	denotes	RMCE
T3145	4395-4404	JJ	denotes	efficient
T3146	4405-4409	RB	denotes	only
T3148	4410-4412	IN	denotes	if
T3149	4413-4416	DT	denotes	the
T3151	4417-4428	NN	denotes	replacement
T3150	4429-4437	NN	denotes	cassette
T3147	4438-4447	VBD	denotes	contained
T3152	4448-4449	DT	denotes	a
T3153	4450-4456	NN	denotes	marker
T3154	4457-4465	VBG	denotes	enabling
T3155	4466-4475	NN	denotes	selection
T3156	4476-4478	IN	denotes	of
T3157	4479-4482	DT	denotes	the
T3159	4483-4490	VBN	denotes	desired
T3158	4491-4502	JJ	denotes	recombinant
T3160	4503-4504	-LRB-	denotes	(
T3161	4504-4505	CD	denotes	7
T3162	4505-4506	,	denotes	,
T3163	4506-4507	CD	denotes	9
T3164	4507-4508	SYM	denotes	–
T3165	4508-4510	CD	denotes	12
T3166	4510-4511	-RRB-	denotes	)
T3167	4511-4512	.	denotes	.
T3168	4512-4668	sentence	denotes	However, interference resulting from expression of the selection marker and the endogenous gene (13) necessitates strategies to remove the selectable gene.
T3169	4513-4520	RB	denotes	However
T3171	4520-4522	,	denotes	,
T3172	4522-4534	NN	denotes	interference
T3173	4535-4544	VBG	denotes	resulting
T3174	4545-4549	IN	denotes	from
T3175	4550-4560	NN	denotes	expression
T3176	4561-4563	IN	denotes	of
T3177	4564-4567	DT	denotes	the
T3179	4568-4577	NN	denotes	selection
T3178	4578-4584	NN	denotes	marker
T3180	4585-4588	CC	denotes	and
T3181	4589-4592	DT	denotes	the
T3183	4593-4603	JJ	denotes	endogenous
T3182	4604-4608	NN	denotes	gene
T3184	4609-4610	-LRB-	denotes	(
T3185	4610-4612	CD	denotes	13
T3186	4612-4613	-RRB-	denotes	)
T3170	4614-4626	VBZ	denotes	necessitates
T3187	4627-4637	NNS	denotes	strategies
T3188	4638-4640	TO	denotes	to
T3189	4641-4647	VB	denotes	remove
T3190	4648-4651	DT	denotes	the
T3192	4652-4662	JJ	denotes	selectable
T3191	4663-4667	NN	denotes	gene
T3193	4667-4668	.	denotes	.
T3194	4668-5012	sentence	denotes	Together, previous studies indicate that an optimal RMCE requires (i) inverted heterologous loxP sites diverging by at least 2 nt to maximize the efficiency of exchange and (ii) an expression cassette enabling both positive selection to identify the initial recombinant and then negative selection to obtain a ‘marker-free’ mutant allele (14).
T3195	4669-4677	RB	denotes	Together
T3197	4677-4679	,	denotes	,
T3198	4679-4687	JJ	denotes	previous
T3199	4688-4695	NNS	denotes	studies
T3196	4696-4704	VBP	denotes	indicate
T3200	4705-4709	IN	denotes	that
T3202	4710-4712	DT	denotes	an
T3204	4713-4720	JJ	denotes	optimal
T3203	4721-4725	NN	denotes	RMCE
T3201	4726-4734	VBZ	denotes	requires
T3205	4735-4736	-LRB-	denotes	(
T3206	4736-4737	LS	denotes	i
T3208	4737-4738	-RRB-	denotes	)
T3209	4739-4747	VBN	denotes	inverted
T3210	4748-4760	JJ	denotes	heterologous
T3211	4761-4765	NN	denotes	loxP
T3207	4766-4771	NNS	denotes	sites
T3212	4772-4781	VBG	denotes	diverging
T3213	4782-4784	IN	denotes	by
T3214	4785-4787	RB	denotes	at
T3216	4788-4793	RBS	denotes	least
T3215	4794-4795	CD	denotes	2
T3217	4796-4798	NN	denotes	nt
T3218	4799-4801	TO	denotes	to
T3219	4802-4810	VB	denotes	maximize
T3220	4811-4814	DT	denotes	the
T3221	4815-4825	NN	denotes	efficiency
T3222	4826-4828	IN	denotes	of
T3223	4829-4837	NN	denotes	exchange
T3224	4838-4841	CC	denotes	and
T3225	4842-4843	-LRB-	denotes	(
T3226	4843-4845	LS	denotes	ii
T3228	4845-4846	-RRB-	denotes	)
T3229	4847-4849	DT	denotes	an
T3230	4850-4860	NN	denotes	expression
T3227	4861-4869	NN	denotes	cassette
T3231	4870-4878	VBG	denotes	enabling
T3232	4879-4883	CC	denotes	both
T3234	4884-4892	JJ	denotes	positive
T3233	4893-4902	NN	denotes	selection
T3235	4903-4905	TO	denotes	to
T3236	4906-4914	VB	denotes	identify
T3237	4915-4918	DT	denotes	the
T3239	4919-4926	JJ	denotes	initial
T3238	4927-4938	JJ	denotes	recombinant
T3240	4939-4942	CC	denotes	and
T3241	4943-4947	RB	denotes	then
T3243	4948-4956	JJ	denotes	negative
T3242	4957-4966	NN	denotes	selection
T3244	4967-4969	TO	denotes	to
T3245	4970-4976	VB	denotes	obtain
T3246	4977-4978	DT	denotes	a
T3248	4979-4980	``	denotes	‘
T3250	4980-4986	NN	denotes	marker
T3251	4986-4987	HYPH	denotes	-
T3249	4987-4991	JJ	denotes	free
T3252	4991-4992	''	denotes	’
T3253	4993-4999	JJ	denotes	mutant
T3247	5000-5006	NN	denotes	allele
T3254	5007-5008	-LRB-	denotes	(
T3255	5008-5010	CD	denotes	14
T3256	5010-5011	-RRB-	denotes	)
T3257	5011-5012	.	denotes	.
T3258	5012-5093	sentence	denotes	Most RMCE experiments have been performed at random sites in somatic cell lines.
T3259	5013-5017	JJS	denotes	Most
T3261	5018-5022	NN	denotes	RMCE
T3260	5023-5034	NNS	denotes	experiments
T3263	5035-5039	VBP	denotes	have
T3264	5040-5044	VBN	denotes	been
T3262	5045-5054	VBN	denotes	performed
T3265	5055-5057	IN	denotes	at
T3266	5058-5064	JJ	denotes	random
T3267	5065-5070	NNS	denotes	sites
T3268	5071-5073	IN	denotes	in
T3269	5074-5081	JJ	denotes	somatic
T3270	5082-5086	NN	denotes	cell
T3271	5087-5092	NNS	denotes	lines
T3272	5092-5093	.	denotes	.
T3273	5093-5303	sentence	denotes	Only a few mutant mice generated by RMCE in ES cells have been reported, but the RMCE systematically introduced a selectable marker (15–17), or, when tested without an incoming marker, proved inefficient (12).
T3274	5094-5098	RB	denotes	Only
T3276	5099-5100	DT	denotes	a
T3277	5101-5104	JJ	denotes	few
T3278	5105-5111	JJ	denotes	mutant
T3275	5112-5116	NNS	denotes	mice
T3280	5117-5126	VBN	denotes	generated
T3281	5127-5129	IN	denotes	by
T3282	5130-5134	NN	denotes	RMCE
T3283	5135-5137	IN	denotes	in
T3284	5138-5140	NN	denotes	ES
T3285	5141-5146	NNS	denotes	cells
T3286	5147-5151	VBP	denotes	have
T3287	5152-5156	VBN	denotes	been
T3279	5157-5165	VBN	denotes	reported
T3288	5165-5167	,	denotes	,
T3289	5167-5170	CC	denotes	but
T3290	5171-5174	DT	denotes	the
T3291	5175-5179	NN	denotes	RMCE
T3293	5180-5194	RB	denotes	systematically
T3292	5195-5205	VBD	denotes	introduced
T3294	5206-5207	DT	denotes	a
T3296	5208-5218	JJ	denotes	selectable
T3295	5219-5225	NN	denotes	marker
T3297	5226-5227	-LRB-	denotes	(
T3298	5227-5232	CD	denotes	15–17
T3299	5232-5233	-RRB-	denotes	)
T3300	5233-5235	,	denotes	,
T3301	5235-5237	CC	denotes	or
T3302	5237-5239	,	denotes	,
T3303	5239-5243	WRB	denotes	when
T3304	5244-5250	VBN	denotes	tested
T3306	5251-5258	IN	denotes	without
T3307	5259-5261	DT	denotes	an
T3309	5262-5270	JJ	denotes	incoming
T3308	5271-5277	NN	denotes	marker
T3310	5277-5279	,	denotes	,
T3305	5279-5285	VBD	denotes	proved
T3311	5286-5297	JJ	denotes	inefficient
T3312	5298-5299	-LRB-	denotes	(
T3313	5299-5301	CD	denotes	12
T3314	5301-5302	-RRB-	denotes	)
T3315	5302-5303	.	denotes	.
T3316	5303-5351	sentence	denotes	A recent report disclosed an additional problem
T3317	5304-5305	DT	denotes	A
T3319	5306-5312	JJ	denotes	recent
T3318	5313-5319	NN	denotes	report
T3320	5320-5329	VBD	denotes	disclosed
T3321	5330-5332	DT	denotes	an
T3323	5333-5343	JJ	denotes	additional
T3322	5344-5351	NN	denotes	problem
T3324	5351-5353	:	denotes	:
T3326	5351-5542	sentence	denotes	: the Hygromycin–Thymidine Kinase fusion gene used most frequently for positive/negative selection in RMCE, leads to mouse sterility, so that exchanges can only be performed in ES cells (16).
T3327	5353-5356	DT	denotes	the
T3329	5357-5367	NN	denotes	Hygromycin
T3331	5367-5368	HYPH	denotes	–
T3330	5368-5377	NN	denotes	Thymidine
T3332	5378-5384	NN	denotes	Kinase
T3333	5385-5391	NN	denotes	fusion
T3328	5392-5396	NN	denotes	gene
T3334	5397-5401	VBN	denotes	used
T3335	5402-5406	RBS	denotes	most
T3336	5407-5417	RB	denotes	frequently
T3337	5418-5421	IN	denotes	for
T3338	5422-5430	JJ	denotes	positive
T3340	5430-5431	HYPH	denotes	/
T3339	5431-5439	JJ	denotes	negative
T3341	5440-5449	NN	denotes	selection
T3342	5450-5452	IN	denotes	in
T3343	5453-5457	NN	denotes	RMCE
T3344	5457-5459	,	denotes	,
T3325	5459-5464	VBZ	denotes	leads
T3345	5465-5467	IN	denotes	to
T3346	5468-5473	NN	denotes	mouse
T3347	5474-5483	NN	denotes	sterility
T3348	5483-5485	,	denotes	,
T3349	5485-5487	IN	denotes	so
T3351	5488-5492	IN	denotes	that
T3352	5493-5502	NNS	denotes	exchanges
T3353	5503-5506	MD	denotes	can
T3354	5507-5511	RB	denotes	only
T3355	5512-5514	VB	denotes	be
T3350	5515-5524	VBN	denotes	performed
T3356	5525-5527	IN	denotes	in
T3357	5528-5530	NN	denotes	ES
T3358	5531-5536	NNS	denotes	cells
T3359	5537-5538	-LRB-	denotes	(
T3360	5538-5540	CD	denotes	16
T3361	5540-5541	-RRB-	denotes	)
T3362	5541-5542	.	denotes	.
T3363	5542-5793	sentence	denotes	The RMCE strategy presented here relies on the integrated use of inverted heterologous loxP sites, a positive/negative selection marker that preserves the germline capacity of ES cells, and the power of mouse genetics to expedite phenotypic analyses.
T3364	5543-5546	DT	denotes	The
T3366	5547-5551	NN	denotes	RMCE
T3365	5552-5560	NN	denotes	strategy
T3368	5561-5570	VBN	denotes	presented
T3369	5571-5575	RB	denotes	here
T3367	5576-5582	VBZ	denotes	relies
T3370	5583-5585	IN	denotes	on
T3371	5586-5589	DT	denotes	the
T3373	5590-5600	VBN	denotes	integrated
T3372	5601-5604	NN	denotes	use
T3374	5605-5607	IN	denotes	of
T3375	5608-5616	VBN	denotes	inverted
T3377	5617-5629	JJ	denotes	heterologous
T3378	5630-5634	NN	denotes	loxP
T3376	5635-5640	NNS	denotes	sites
T3379	5640-5642	,	denotes	,
T3380	5642-5643	DT	denotes	a
T3382	5644-5652	JJ	denotes	positive
T3384	5652-5653	HYPH	denotes	/
T3383	5653-5661	JJ	denotes	negative
T3385	5662-5671	NN	denotes	selection
T3381	5672-5678	NN	denotes	marker
T3386	5679-5683	WDT	denotes	that
T3387	5684-5693	VBZ	denotes	preserves
T3388	5694-5697	DT	denotes	the
T3390	5698-5706	NN	denotes	germline
T3389	5707-5715	NN	denotes	capacity
T3391	5716-5718	IN	denotes	of
T3392	5719-5721	NN	denotes	ES
T3393	5722-5727	NNS	denotes	cells
T3394	5727-5729	,	denotes	,
T3395	5729-5732	CC	denotes	and
T3396	5733-5736	DT	denotes	the
T3397	5737-5742	NN	denotes	power
T3398	5743-5745	IN	denotes	of
T3399	5746-5751	NN	denotes	mouse
T3400	5752-5760	NN	denotes	genetics
T3401	5761-5763	TO	denotes	to
T3402	5764-5772	VB	denotes	expedite
T3403	5773-5783	JJ	denotes	phenotypic
T3404	5784-5792	NNS	denotes	analyses
T3405	5792-5793	.	denotes	.
T3406	5793-6027	sentence	denotes	We show that our approach enables efficient targeting of marker-free mutations at the p53 locus in ES cells to generate mutant mice, but more importantly, it is Adapted for targeting in Somatic cells to Accelerate Phenotyping (ASAP).
T3407	5794-5796	PRP	denotes	We
T3408	5797-5801	VBP	denotes	show
T3409	5802-5806	IN	denotes	that
T3411	5807-5810	PRP$	denotes	our
T3412	5811-5819	NN	denotes	approach
T3410	5820-5827	VBZ	denotes	enables
T3413	5828-5837	JJ	denotes	efficient
T3414	5838-5847	NN	denotes	targeting
T3415	5848-5850	IN	denotes	of
T3416	5851-5857	JJ	denotes	marker
T3418	5857-5858	HYPH	denotes	-
T3417	5858-5862	JJ	denotes	free
T3419	5863-5872	NNS	denotes	mutations
T3420	5873-5875	IN	denotes	at
T3421	5876-5879	DT	denotes	the
T3423	5880-5883	NN	denotes	p53
T3422	5884-5889	NN	denotes	locus
T3424	5890-5892	IN	denotes	in
T3425	5893-5895	NN	denotes	ES
T3426	5896-5901	NNS	denotes	cells
T3427	5902-5904	TO	denotes	to
T3428	5905-5913	VB	denotes	generate
T3429	5914-5920	JJ	denotes	mutant
T3430	5921-5925	NNS	denotes	mice
T3431	5925-5927	,	denotes	,
T3432	5927-5930	CC	denotes	but
T3433	5931-5935	RBR	denotes	more
T3434	5936-5947	RB	denotes	importantly
T3436	5947-5949	,	denotes	,
T3437	5949-5951	PRP	denotes	it
T3438	5952-5954	VBZ	denotes	is
T3435	5955-5962	VBN	denotes	Adapted
T3439	5963-5966	IN	denotes	for
T3440	5967-5976	VBG	denotes	targeting
T3441	5977-5979	IN	denotes	in
T3442	5980-5987	JJ	denotes	Somatic
T3443	5988-5993	NNS	denotes	cells
T3444	5994-5996	TO	denotes	to
T3445	5997-6007	VB	denotes	Accelerate
T3446	6008-6019	NN	denotes	Phenotyping
T3447	6020-6021	-LRB-	denotes	(
T3448	6021-6025	NN	denotes	ASAP
T3449	6025-6026	-RRB-	denotes	)
T3450	6026-6027	.	denotes	.
T3451	6027-6126	sentence	denotes	Because it relies on very general principles, RMCE-ASAP could be applied to any locus of interest.
T3452	6028-6035	IN	denotes	Because
T3454	6036-6038	PRP	denotes	it
T3453	6039-6045	VBZ	denotes	relies
T3456	6046-6048	IN	denotes	on
T3457	6049-6053	RB	denotes	very
T3459	6054-6061	JJ	denotes	general
T3458	6062-6072	NNS	denotes	principles
T3460	6072-6074	,	denotes	,
T3461	6074-6078	NN	denotes	RMCE
T3463	6078-6079	HYPH	denotes	-
T3462	6079-6083	NN	denotes	ASAP
T3464	6084-6089	MD	denotes	could
T3465	6090-6092	VB	denotes	be
T3455	6093-6100	VBN	denotes	applied
T3466	6101-6103	IN	denotes	to
T3467	6104-6107	DT	denotes	any
T3468	6108-6113	NN	denotes	locus
T3469	6114-6116	IN	denotes	of
T3470	6117-6125	NN	denotes	interest
T3471	6125-6126	.	denotes	.
T4611	6151-6160	NN	denotes	Targeting
T4612	6161-6170	NN	denotes	construct
T4613	6171-6174	IN	denotes	for
T4614	6175-6176	DT	denotes	a
T4616	6177-6180	NN	denotes	p53
T4617	6181-6185	NN	denotes	RMCE
T4619	6185-6186	HYPH	denotes	-
T4618	6186-6191	JJ	denotes	ready
T4615	6192-6197	NN	denotes	locus
T4620	6197-6311	sentence	denotes	Details for plasmid construction are available upon request owing to space limitations mandating a brief outline.
T4621	6198-6205	NNS	denotes	Details
T4623	6206-6209	IN	denotes	for
T4624	6210-6217	NN	denotes	plasmid
T4625	6218-6230	NN	denotes	construction
T4622	6231-6234	VBP	denotes	are
T4626	6235-6244	JJ	denotes	available
T4627	6245-6249	IN	denotes	upon
T4628	6250-6257	NN	denotes	request
T4629	6258-6263	VBG	denotes	owing
T4630	6264-6266	IN	denotes	to
T4631	6267-6272	NN	denotes	space
T4632	6273-6284	NNS	denotes	limitations
T4633	6285-6294	VBG	denotes	mandating
T4634	6295-6296	DT	denotes	a
T4636	6297-6302	JJ	denotes	brief
T4635	6303-6310	NN	denotes	outline
T4637	6310-6311	.	denotes	.
T4638	6311-6457	sentence	denotes	We started from a plasmid L3-1L containing heterologous loxP sites (L3 is the mutant loxP257 recently described (14), 1L is an inverted WT loxP).
T4639	6312-6314	PRP	denotes	We
T4640	6315-6322	VBD	denotes	started
T4641	6323-6327	IN	denotes	from
T4642	6328-6329	DT	denotes	a
T4644	6330-6337	NN	denotes	plasmid
T4645	6338-6340	NN	denotes	L3
T4646	6340-6341	HYPH	denotes	-
T4643	6341-6343	NN	denotes	1L
T4647	6344-6354	VBG	denotes	containing
T4648	6355-6367	JJ	denotes	heterologous
T4650	6368-6372	NN	denotes	loxP
T4649	6373-6378	NNS	denotes	sites
T4651	6379-6380	-LRB-	denotes	(
T4653	6380-6382	NN	denotes	L3
T4654	6383-6385	VBZ	denotes	is
T4655	6386-6389	DT	denotes	the
T4657	6390-6396	JJ	denotes	mutant
T4656	6397-6404	NN	denotes	loxP257
T4658	6405-6413	RB	denotes	recently
T4659	6414-6423	VBN	denotes	described
T4660	6424-6425	-LRB-	denotes	(
T4661	6425-6427	CD	denotes	14
T4662	6427-6428	-RRB-	denotes	)
T4663	6428-6430	,	denotes	,
T4664	6430-6432	NN	denotes	1L
T4652	6433-6435	VBZ	denotes	is
T4665	6436-6438	DT	denotes	an
T4667	6439-6447	VBN	denotes	inverted
T4668	6448-6450	NN	denotes	WT
T4666	6451-6455	NN	denotes	loxP
T4669	6455-6456	-RRB-	denotes	)
T4670	6456-6457	.	denotes	.
T4671	6457-6597	sentence	denotes	The WT loxP and loxP257 differ in their spacer sequences: the spacer sequence is 5′-ATGTATGC-3′ for WT loxP and 5′-AAGTCTCC-3′ for loxP257.
T4672	6458-6461	DT	denotes	The
T4674	6462-6464	NN	denotes	WT
T4673	6465-6469	NN	denotes	loxP
T4676	6470-6473	CC	denotes	and
T4677	6474-6481	NN	denotes	loxP257
T4675	6482-6488	VBP	denotes	differ
T4679	6489-6491	IN	denotes	in
T4680	6492-6497	PRP$	denotes	their
T4682	6498-6504	NN	denotes	spacer
T4681	6505-6514	NNS	denotes	sequences
T4683	6514-6516	:	denotes	:
T4684	6516-6519	DT	denotes	the
T4686	6520-6526	NN	denotes	spacer
T4685	6527-6535	NN	denotes	sequence
T4678	6536-6538	VBZ	denotes	is
T4687	6539-6540	CD	denotes	5
T4688	6540-6541	SYM	denotes	′
T4689	6541-6542	HYPH	denotes	-
T4690	6542-6550	JJ	denotes	ATGTATGC
T4691	6550-6551	HYPH	denotes	-
T4692	6551-6552	CD	denotes	3
T4693	6552-6553	SYM	denotes	′
T4694	6554-6557	IN	denotes	for
T4695	6558-6560	NN	denotes	WT
T4696	6561-6565	NN	denotes	loxP
T4697	6566-6569	CC	denotes	and
T4698	6570-6571	CD	denotes	5
T4700	6571-6572	SYM	denotes	′
T4701	6572-6573	HYPH	denotes	-
T4699	6573-6581	NN	denotes	AAGTCTCC
T4702	6581-6582	HYPH	denotes	-
T4703	6582-6583	CD	denotes	3
T4704	6583-6584	SYM	denotes	′
T4705	6585-6588	IN	denotes	for
T4706	6589-6596	NN	denotes	loxP257
T4707	6596-6597	.	denotes	.
T4708	6597-6901	sentence	denotes	The three mutations in the loxP257 spacer sequence prevent it to recombine with WT loxP, ensuring efficient RMCE in several cell lines: accurate RMCE with these loxP sites occurred with an average frequency of 81% at two loci in CHO cells and an average frequency of 69% at four loci in Hela cells (14).
T4709	6598-6601	DT	denotes	The
T4711	6602-6607	CD	denotes	three
T4710	6608-6617	NNS	denotes	mutations
T4713	6618-6620	IN	denotes	in
T4714	6621-6624	DT	denotes	the
T4716	6625-6632	NN	denotes	loxP257
T4717	6633-6639	NN	denotes	spacer
T4715	6640-6648	NN	denotes	sequence
T4712	6649-6656	VBP	denotes	prevent
T4719	6657-6659	PRP	denotes	it
T4721	6660-6662	TO	denotes	to
T4720	6663-6672	VB	denotes	recombine
T4722	6673-6677	IN	denotes	with
T4723	6678-6680	NN	denotes	WT
T4724	6681-6685	NN	denotes	loxP
T4725	6685-6687	,	denotes	,
T4726	6687-6695	VBG	denotes	ensuring
T4727	6696-6705	JJ	denotes	efficient
T4728	6706-6710	NN	denotes	RMCE
T4729	6711-6713	IN	denotes	in
T4730	6714-6721	JJ	denotes	several
T4732	6722-6726	NN	denotes	cell
T4731	6727-6732	NNS	denotes	lines
T4733	6732-6734	:	denotes	:
T4734	6734-6742	JJ	denotes	accurate
T4735	6743-6747	NN	denotes	RMCE
T4736	6748-6752	IN	denotes	with
T4737	6753-6758	DT	denotes	these
T4739	6759-6763	NN	denotes	loxP
T4738	6764-6769	NNS	denotes	sites
T4718	6770-6778	VBD	denotes	occurred
T4740	6779-6783	IN	denotes	with
T4741	6784-6786	DT	denotes	an
T4743	6787-6794	JJ	denotes	average
T4742	6795-6804	NN	denotes	frequency
T4744	6805-6807	IN	denotes	of
T4745	6808-6810	CD	denotes	81
T4746	6810-6811	NN	denotes	%
T4747	6812-6814	IN	denotes	at
T4748	6815-6818	CD	denotes	two
T4749	6819-6823	NNS	denotes	loci
T4750	6824-6826	IN	denotes	in
T4751	6827-6830	NN	denotes	CHO
T4752	6831-6836	NNS	denotes	cells
T4753	6837-6840	CC	denotes	and
T4754	6841-6843	DT	denotes	an
T4756	6844-6851	JJ	denotes	average
T4755	6852-6861	NN	denotes	frequency
T4757	6862-6864	IN	denotes	of
T4758	6865-6867	CD	denotes	69
T4759	6867-6868	NN	denotes	%
T4760	6869-6871	IN	denotes	at
T4761	6872-6876	CD	denotes	four
T4762	6877-6881	NNS	denotes	loci
T4763	6882-6884	IN	denotes	in
T4764	6885-6889	NN	denotes	Hela
T4765	6890-6895	NNS	denotes	cells
T4766	6896-6897	-LRB-	denotes	(
T4767	6897-6899	CD	denotes	14
T4768	6899-6900	-RRB-	denotes	)
T4769	6900-6901	.	denotes	.
T4770	6901-7025	sentence	denotes	The L3-1L plasmid was first modified to include a ClaI and a FseI site between the LoxP sites, leading to plasmid L3-CF-1L.
T4771	6902-6905	DT	denotes	The
T4773	6906-6908	NN	denotes	L3
T4775	6908-6909	HYPH	denotes	-
T4774	6909-6911	NN	denotes	1L
T4772	6912-6919	NN	denotes	plasmid
T4777	6920-6923	VBD	denotes	was
T4778	6924-6929	RB	denotes	first
T4776	6930-6938	VBN	denotes	modified
T4779	6939-6941	TO	denotes	to
T4780	6942-6949	VB	denotes	include
T4781	6950-6951	DT	denotes	a
T4782	6952-6956	NN	denotes	ClaI
T4783	6957-6960	CC	denotes	and
T4784	6961-6962	DT	denotes	a
T4786	6963-6967	NN	denotes	FseI
T4785	6968-6972	NN	denotes	site
T4787	6973-6980	IN	denotes	between
T4788	6981-6984	DT	denotes	the
T4790	6985-6989	NN	denotes	LoxP
T4789	6990-6995	NNS	denotes	sites
T4791	6995-6997	,	denotes	,
T4792	6997-7004	VBG	denotes	leading
T4793	7005-7007	IN	denotes	to
T4794	7008-7015	NN	denotes	plasmid
T4796	7016-7018	NN	denotes	L3
T4797	7018-7019	HYPH	denotes	-
T4798	7019-7021	NN	denotes	CF
T4799	7021-7022	HYPH	denotes	-
T4795	7022-7024	NN	denotes	1L
T4800	7024-7025	.	denotes	.
T4801	7025-7287	sentence	denotes	We next modified a puroΔTK plasmid (YTC37, a kind gift from A. Bradley) by using oligonucleotides to destroy a NotI site downstream of the puroΔTK gene and introduce a NotI site upstream, and a FseI site downstream of the gene (leading to plasmid CN-PuroΔTK-F).
T4802	7026-7028	PRP	denotes	We
T4804	7029-7033	RB	denotes	next
T4803	7034-7042	VBD	denotes	modified
T4805	7043-7044	DT	denotes	a
T4807	7045-7052	NN	denotes	puroΔTK
T4806	7053-7060	NN	denotes	plasmid
T4808	7061-7062	-LRB-	denotes	(
T4809	7062-7067	NN	denotes	YTC37
T4810	7067-7069	,	denotes	,
T4811	7069-7070	DT	denotes	a
T4813	7071-7075	NN	denotes	kind
T4812	7076-7080	NN	denotes	gift
T4814	7081-7085	IN	denotes	from
T4815	7086-7088	NNP	denotes	A.
T4816	7089-7096	NNP	denotes	Bradley
T4817	7096-7097	-RRB-	denotes	)
T4818	7098-7100	IN	denotes	by
T4819	7101-7106	VBG	denotes	using
T4820	7107-7123	NNS	denotes	oligonucleotides
T4821	7124-7126	TO	denotes	to
T4822	7127-7134	VB	denotes	destroy
T4823	7135-7136	DT	denotes	a
T4825	7137-7141	NN	denotes	NotI
T4824	7142-7146	NN	denotes	site
T4826	7147-7157	RB	denotes	downstream
T4827	7158-7160	IN	denotes	of
T4828	7161-7164	DT	denotes	the
T4830	7165-7172	NN	denotes	puroΔTK
T4829	7173-7177	NN	denotes	gene
T4831	7178-7181	CC	denotes	and
T4832	7182-7191	VB	denotes	introduce
T4833	7192-7193	DT	denotes	a
T4835	7194-7198	NN	denotes	NotI
T4834	7199-7203	NN	denotes	site
T4836	7204-7212	RB	denotes	upstream
T4837	7212-7214	,	denotes	,
T4838	7214-7217	CC	denotes	and
T4839	7218-7219	DT	denotes	a
T4841	7220-7224	NN	denotes	FseI
T4840	7225-7229	NN	denotes	site
T4842	7230-7240	RB	denotes	downstream
T4843	7241-7243	IN	denotes	of
T4844	7244-7247	DT	denotes	the
T4845	7248-7252	NN	denotes	gene
T4846	7253-7254	-LRB-	denotes	(
T4847	7254-7261	VBG	denotes	leading
T4848	7262-7264	IN	denotes	to
T4849	7265-7272	NN	denotes	plasmid
T4851	7273-7275	NN	denotes	CN
T4852	7275-7276	HYPH	denotes	-
T4853	7276-7283	NN	denotes	PuroΔTK
T4854	7283-7284	HYPH	denotes	-
T4850	7284-7285	NN	denotes	F
T4855	7285-7286	-RRB-	denotes	)
T4856	7286-7287	.	denotes	.
T4857	7287-7535	sentence	denotes	Next, a PmlI-MfeI 6.3 kb fragment from Trp53 was subcloned in a modified pBluescript KSII+ (pBS, Stratagene), and the resulting plasmid was digested with SwaI to introduce an EagI site, leading to p53PmlEag, a plasmid containing exons 2–11 of p53.
T4858	7288-7292	RB	denotes	Next
T4860	7292-7294	,	denotes	,
T4861	7294-7295	DT	denotes	a
T4863	7296-7300	NN	denotes	PmlI
T4865	7300-7301	HYPH	denotes	-
T4864	7301-7305	NN	denotes	MfeI
T4866	7306-7309	CD	denotes	6.3
T4867	7310-7312	NN	denotes	kb
T4862	7313-7321	NN	denotes	fragment
T4868	7322-7326	IN	denotes	from
T4869	7327-7332	NN	denotes	Trp53
T4870	7333-7336	VBD	denotes	was
T4859	7337-7346	VBN	denotes	subcloned
T4871	7347-7349	IN	denotes	in
T4872	7350-7351	DT	denotes	a
T4874	7352-7360	VBN	denotes	modified
T4875	7361-7372	NN	denotes	pBluescript
T4873	7373-7378	NN	denotes	KSII+
T4876	7379-7380	-LRB-	denotes	(
T4877	7380-7383	NN	denotes	pBS
T4878	7383-7385	,	denotes	,
T4879	7385-7395	NNP	denotes	Stratagene
T4880	7395-7396	-RRB-	denotes	)
T4881	7396-7398	,	denotes	,
T4882	7398-7401	CC	denotes	and
T4883	7402-7405	DT	denotes	the
T4885	7406-7415	VBG	denotes	resulting
T4884	7416-7423	NN	denotes	plasmid
T4887	7424-7427	VBD	denotes	was
T4886	7428-7436	VBN	denotes	digested
T4888	7437-7441	IN	denotes	with
T4889	7442-7446	NN	denotes	SwaI
T4890	7447-7449	TO	denotes	to
T4891	7450-7459	VB	denotes	introduce
T4892	7460-7462	DT	denotes	an
T4894	7463-7467	NN	denotes	EagI
T4893	7468-7472	NN	denotes	site
T4895	7472-7474	,	denotes	,
T4896	7474-7481	VBG	denotes	leading
T4897	7482-7484	IN	denotes	to
T4898	7485-7494	NN	denotes	p53PmlEag
T4899	7494-7496	,	denotes	,
T4900	7496-7497	DT	denotes	a
T4901	7498-7505	NN	denotes	plasmid
T4902	7506-7516	VBG	denotes	containing
T4903	7517-7522	NNS	denotes	exons
T4905	7523-7524	CD	denotes	2
T4906	7524-7525	HYPH	denotes	–
T4904	7525-7527	CD	denotes	11
T4907	7528-7530	IN	denotes	of
T4908	7531-7534	NN	denotes	p53
T4909	7534-7535	.	denotes	.
T4910	7535-7780	sentence	denotes	We then inserted a 5.5 kb ClaI-EagI fragment from p53PmlEag in plasmid CN-PuroΔTK-F digested by ClaI and NotI, and inserted the resulting fragment between the loxP sites of L3-CF-1L by ClaI and FseI digestion, leading to L3-p53PmlEagPuroΔTK-1L.
T4911	7536-7538	PRP	denotes	We
T4913	7539-7543	RB	denotes	then
T4912	7544-7552	VBD	denotes	inserted
T4914	7553-7554	DT	denotes	a
T4916	7555-7558	CD	denotes	5.5
T4917	7559-7561	NN	denotes	kb
T4918	7562-7566	NN	denotes	ClaI
T4920	7566-7567	HYPH	denotes	-
T4919	7567-7571	NN	denotes	EagI
T4915	7572-7580	NN	denotes	fragment
T4921	7581-7585	IN	denotes	from
T4922	7586-7595	NN	denotes	p53PmlEag
T4923	7596-7598	IN	denotes	in
T4924	7599-7606	NN	denotes	plasmid
T4926	7607-7609	NN	denotes	CN
T4927	7609-7610	HYPH	denotes	-
T4928	7610-7617	NN	denotes	PuroΔTK
T4929	7617-7618	HYPH	denotes	-
T4925	7618-7619	NN	denotes	F
T4930	7620-7628	VBN	denotes	digested
T4931	7629-7631	IN	denotes	by
T4932	7632-7636	NN	denotes	ClaI
T4933	7637-7640	CC	denotes	and
T4934	7641-7645	NN	denotes	NotI
T4935	7645-7647	,	denotes	,
T4936	7647-7650	CC	denotes	and
T4937	7651-7659	VBD	denotes	inserted
T4938	7660-7663	DT	denotes	the
T4940	7664-7673	VBG	denotes	resulting
T4939	7674-7682	NN	denotes	fragment
T4941	7683-7690	IN	denotes	between
T4942	7691-7694	DT	denotes	the
T4944	7695-7699	NN	denotes	loxP
T4943	7700-7705	NNS	denotes	sites
T4945	7706-7708	IN	denotes	of
T4946	7709-7711	NN	denotes	L3
T4948	7711-7712	HYPH	denotes	-
T4949	7712-7714	NN	denotes	CF
T4950	7714-7715	HYPH	denotes	-
T4947	7715-7717	NN	denotes	1L
T4951	7718-7720	IN	denotes	by
T4952	7721-7725	NN	denotes	ClaI
T4954	7726-7729	CC	denotes	and
T4955	7730-7734	NN	denotes	FseI
T4953	7735-7744	NN	denotes	digestion
T4956	7744-7746	,	denotes	,
T4957	7746-7753	VBG	denotes	leading
T4958	7754-7756	IN	denotes	to
T4959	7757-7759	NN	denotes	L3
T4961	7759-7760	HYPH	denotes	-
T4962	7760-7776	NN	denotes	p53PmlEagPuroΔTK
T4963	7776-7777	HYPH	denotes	-
T4960	7777-7779	NN	denotes	1L
T4964	7779-7780	.	denotes	.
T4965	7780-8370	sentence	denotes	We next engineered a plasmid containing the region for 3′-homology downstream of the p53 gene and the DTA gene in two steps: (i) we performed a three-way ligation between a modified pBS digested by HindIII and NotI, a HindIII-EcoRI fragment from Trp53 for 3′homology and an EcoRI-NotI fragment containing the DTA gene, from plasmid pgkdtabpa (kind gift of P. Soriano), leading to plasmid 3′ + DTA and (ii) because the Bsu36I-EcoRI region downstream of p53 contains repetitive sequences (F. Toledo and G. M. Wahl, unpublished data), we later deleted this region, to obtain plasmid 3′ + DTA.
T4966	7781-7783	PRP	denotes	We
T4968	7784-7788	RB	denotes	next
T4967	7789-7799	VBD	denotes	engineered
T4970	7800-7801	DT	denotes	a
T4971	7802-7809	NN	denotes	plasmid
T4972	7810-7820	VBG	denotes	containing
T4973	7821-7824	DT	denotes	the
T4974	7825-7831	NN	denotes	region
T4975	7832-7835	IN	denotes	for
T4976	7836-7837	CD	denotes	3
T4977	7837-7838	SYM	denotes	′
T4978	7838-7839	SYM	denotes	-
T4979	7839-7847	JJ	denotes	homology
T4980	7848-7858	RB	denotes	downstream
T4981	7859-7861	IN	denotes	of
T4982	7862-7865	DT	denotes	the
T4984	7866-7869	NN	denotes	p53
T4983	7870-7874	NN	denotes	gene
T4985	7875-7878	CC	denotes	and
T4986	7879-7882	DT	denotes	the
T4988	7883-7886	NN	denotes	DTA
T4987	7887-7891	NN	denotes	gene
T4989	7892-7894	IN	denotes	in
T4990	7895-7898	CD	denotes	two
T4991	7899-7904	NNS	denotes	steps
T4992	7904-7906	:	denotes	:
T4993	7906-7907	-LRB-	denotes	(
T4994	7907-7908	LS	denotes	i
T4995	7908-7909	-RRB-	denotes	)
T4996	7910-7912	PRP	denotes	we
T4969	7913-7922	VBD	denotes	performed
T4997	7923-7924	DT	denotes	a
T4999	7925-7930	CD	denotes	three
T5001	7930-7931	HYPH	denotes	-
T5000	7931-7934	NN	denotes	way
T4998	7935-7943	NN	denotes	ligation
T5002	7944-7951	IN	denotes	between
T5003	7952-7953	DT	denotes	a
T5005	7954-7962	VBN	denotes	modified
T5004	7963-7966	NN	denotes	pBS
T5006	7967-7975	VBN	denotes	digested
T5007	7976-7978	IN	denotes	by
T5008	7979-7986	NN	denotes	HindIII
T5009	7987-7990	CC	denotes	and
T5010	7991-7995	NN	denotes	NotI
T5011	7995-7997	,	denotes	,
T5012	7997-7998	DT	denotes	a
T5014	7999-8006	NN	denotes	HindIII
T5016	8006-8007	HYPH	denotes	-
T5015	8007-8012	NN	denotes	EcoRI
T5013	8013-8021	NN	denotes	fragment
T5017	8022-8026	IN	denotes	from
T5018	8027-8032	NN	denotes	Trp53
T5019	8033-8036	IN	denotes	for
T5020	8037-8038	CD	denotes	3
T5022	8038-8039	SYM	denotes	′
T5021	8039-8047	NN	denotes	homology
T5023	8048-8051	CC	denotes	and
T5024	8052-8054	DT	denotes	an
T5026	8055-8060	NN	denotes	EcoRI
T5028	8060-8061	HYPH	denotes	-
T5027	8061-8065	NN	denotes	NotI
T5025	8066-8074	NN	denotes	fragment
T5029	8075-8085	VBG	denotes	containing
T5030	8086-8089	DT	denotes	the
T5032	8090-8093	NN	denotes	DTA
T5031	8094-8098	NN	denotes	gene
T5033	8098-8100	,	denotes	,
T5034	8100-8104	IN	denotes	from
T5035	8105-8112	NN	denotes	plasmid
T5036	8113-8122	NN	denotes	pgkdtabpa
T5037	8123-8124	-LRB-	denotes	(
T5038	8124-8128	JJ	denotes	kind
T5039	8129-8133	NN	denotes	gift
T5040	8134-8136	IN	denotes	of
T5041	8137-8139	NNP	denotes	P.
T5042	8140-8147	NNP	denotes	Soriano
T5043	8147-8148	-RRB-	denotes	)
T5044	8148-8150	,	denotes	,
T5045	8150-8157	VBG	denotes	leading
T5046	8158-8160	IN	denotes	to
T5047	8161-8168	NN	denotes	plasmid
T5049	8169-8170	CD	denotes	3
T5050	8170-8171	SYM	denotes	′
T5051	8172-8173	SYM	denotes	+
T5048	8174-8177	NN	denotes	DTA
T5052	8178-8181	CC	denotes	and
T5053	8182-8183	-LRB-	denotes	(
T5054	8183-8185	LS	denotes	ii
T5056	8185-8186	-RRB-	denotes	)
T5057	8187-8194	IN	denotes	because
T5059	8195-8198	DT	denotes	the
T5061	8199-8205	NN	denotes	Bsu36I
T5063	8205-8206	HYPH	denotes	-
T5062	8206-8211	NN	denotes	EcoRI
T5060	8212-8218	NN	denotes	region
T5064	8219-8229	RB	denotes	downstream
T5065	8230-8232	IN	denotes	of
T5066	8233-8236	NN	denotes	p53
T5058	8237-8245	VBZ	denotes	contains
T5067	8246-8256	JJ	denotes	repetitive
T5068	8257-8266	NNS	denotes	sequences
T5069	8267-8268	-LRB-	denotes	(
T5070	8268-8270	NNP	denotes	F.
T5071	8271-8277	NNP	denotes	Toledo
T5072	8278-8281	CC	denotes	and
T5073	8282-8284	NNP	denotes	G.
T5074	8285-8287	NNP	denotes	M.
T5075	8288-8292	NNP	denotes	Wahl
T5076	8292-8294	,	denotes	,
T5077	8294-8305	JJ	denotes	unpublished
T5078	8306-8310	NNS	denotes	data
T5079	8310-8311	-RRB-	denotes	)
T5080	8311-8313	,	denotes	,
T5081	8313-8315	PRP	denotes	we
T5082	8316-8321	RB	denotes	later
T5055	8322-8329	VBD	denotes	deleted
T5083	8330-8334	DT	denotes	this
T5084	8335-8341	NN	denotes	region
T5085	8341-8343	,	denotes	,
T5086	8343-8345	TO	denotes	to
T5087	8346-8352	VB	denotes	obtain
T5088	8353-8360	NN	denotes	plasmid
T5090	8361-8362	CD	denotes	3
T5091	8362-8363	SYM	denotes	′
T5092	8364-8365	SYM	denotes	+
T5089	8366-8369	NN	denotes	DTA
T5093	8369-8370	.	denotes	.
T5094	8370-8493	sentence	denotes	The 5′ homology consists of a 3.4 kb-long BamHI-PmlI fragment from intron 1 of p53 cloned in a modified pBS (plasmid p5′).
T5095	8371-8374	DT	denotes	The
T5097	8375-8376	CD	denotes	5
T5098	8376-8377	SYM	denotes	′
T5096	8378-8386	NN	denotes	homology
T5099	8387-8395	VBZ	denotes	consists
T5100	8396-8398	IN	denotes	of
T5101	8399-8400	DT	denotes	a
T5103	8401-8404	CD	denotes	3.4
T5104	8405-8407	NN	denotes	kb
T5106	8407-8408	HYPH	denotes	-
T5105	8408-8412	JJ	denotes	long
T5107	8413-8418	NN	denotes	BamHI
T5109	8418-8419	HYPH	denotes	-
T5108	8419-8423	NN	denotes	PmlI
T5102	8424-8432	NN	denotes	fragment
T5110	8433-8437	IN	denotes	from
T5111	8438-8444	NN	denotes	intron
T5112	8445-8446	CD	denotes	1
T5113	8447-8449	IN	denotes	of
T5114	8450-8453	NN	denotes	p53
T5115	8454-8460	VBN	denotes	cloned
T5116	8461-8463	IN	denotes	in
T5117	8464-8465	DT	denotes	a
T5119	8466-8474	VBN	denotes	modified
T5118	8475-8478	NN	denotes	pBS
T5120	8479-8480	-LRB-	denotes	(
T5121	8480-8487	NN	denotes	plasmid
T5122	8488-8490	NN	denotes	p5
T5123	8490-8491	SYM	denotes	′
T5124	8491-8492	-RRB-	denotes	)
T5125	8492-8493	.	denotes	.
T5126	8493-8634	sentence	denotes	Finally, appropriate fragments from plasmids p5′, L3-p53PmlEagPuroΔTK-1L, and 3′ + DTA were assembled in a modified pSP72 plasmid (Promega).
T5127	8494-8501	RB	denotes	Finally
T5129	8501-8503	,	denotes	,
T5130	8503-8514	JJ	denotes	appropriate
T5131	8515-8524	NNS	denotes	fragments
T5132	8525-8529	IN	denotes	from
T5133	8530-8538	NNS	denotes	plasmids
T5134	8539-8541	NN	denotes	p5
T5135	8541-8542	SYM	denotes	′
T5136	8542-8544	,	denotes	,
T5137	8544-8546	NN	denotes	L3
T5139	8546-8547	HYPH	denotes	-
T5140	8547-8563	NN	denotes	p53PmlEagPuroΔTK
T5141	8563-8564	HYPH	denotes	-
T5138	8564-8566	NN	denotes	1L
T5142	8566-8568	,	denotes	,
T5143	8568-8571	CC	denotes	and
T5144	8572-8573	CD	denotes	3
T5146	8573-8574	SYM	denotes	′
T5147	8575-8576	SYM	denotes	+
T5145	8577-8580	NN	denotes	DTA
T5148	8581-8585	VBD	denotes	were
T5128	8586-8595	VBN	denotes	assembled
T5149	8596-8598	IN	denotes	in
T5150	8599-8600	DT	denotes	a
T5152	8601-8609	VBN	denotes	modified
T5153	8610-8615	NN	denotes	pSP72
T5151	8616-8623	NN	denotes	plasmid
T5154	8624-8625	-LRB-	denotes	(
T5155	8625-8632	NNP	denotes	Promega
T5156	8632-8633	-RRB-	denotes	)
T5157	8633-8634	.	denotes	.
T5158	8634-8880	sentence	denotes	Plasmid Flox, the resulting targeting construct, was verified by restriction analysis, then sequenced using 30 primers chosen to precisely verify all p53 coding sequences, all exon–intron junctions and the sequences at and around the loxP sites.
T5159	8635-8642	NN	denotes	Plasmid
T5160	8643-8647	NN	denotes	Flox
T5162	8647-8649	,	denotes	,
T5163	8649-8652	DT	denotes	the
T5165	8653-8662	VBG	denotes	resulting
T5166	8663-8672	NN	denotes	targeting
T5164	8673-8682	NN	denotes	construct
T5167	8682-8684	,	denotes	,
T5168	8684-8687	VBD	denotes	was
T5161	8688-8696	VBN	denotes	verified
T5169	8697-8699	IN	denotes	by
T5170	8700-8711	NN	denotes	restriction
T5171	8712-8720	NN	denotes	analysis
T5172	8720-8722	,	denotes	,
T5173	8722-8726	RB	denotes	then
T5174	8727-8736	VBN	denotes	sequenced
T5175	8737-8742	VBG	denotes	using
T5176	8743-8745	CD	denotes	30
T5177	8746-8753	NNS	denotes	primers
T5178	8754-8760	VBN	denotes	chosen
T5179	8761-8763	TO	denotes	to
T5181	8764-8773	RB	denotes	precisely
T5180	8774-8780	VB	denotes	verify
T5182	8781-8784	DT	denotes	all
T5184	8785-8788	NN	denotes	p53
T5185	8789-8795	NN	denotes	coding
T5183	8796-8805	NNS	denotes	sequences
T5186	8805-8807	,	denotes	,
T5187	8807-8810	DT	denotes	all
T5189	8811-8815	NN	denotes	exon
T5191	8815-8816	HYPH	denotes	–
T5190	8816-8822	NN	denotes	intron
T5188	8823-8832	NNS	denotes	junctions
T5192	8833-8836	CC	denotes	and
T5193	8837-8840	DT	denotes	the
T5194	8841-8850	NNS	denotes	sequences
T5195	8851-8853	IN	denotes	at
T5196	8854-8857	CC	denotes	and
T5197	8858-8864	IN	denotes	around
T5198	8865-8868	DT	denotes	the
T5200	8869-8873	NN	denotes	loxP
T5199	8874-8879	NNS	denotes	sites
T5201	8879-8880	.	denotes	.
T5752	8882-8890	NN	denotes	Exchange
T5753	8891-8901	NNS	denotes	constructs
T5754	8901-8903	:	denotes	:
T5755	8903-8909	VBG	denotes	making
T5756	8910-8913	DT	denotes	the
T5757	8914-8920	NN	denotes	p53GFP
T5759	8921-8924	CC	denotes	and
T5760	8925-8928	NN	denotes	p53
T5762	8928-8929	NN	denotes	Δ
T5761	8929-8933	NN	denotes	PGFP
T5758	8934-8942	NNS	denotes	plasmids
T5763	8942-9166	sentence	denotes	To make a p53-GFP fusion protein, we first subcloned a SacII-HindIII fragment of the p53 locus (corresponding to part of exon 10 to sequences downstream of the gene) into pBS, then mutated the HindIII site into a FseI site.
T5764	8943-8945	TO	denotes	To
T5765	8946-8950	VB	denotes	make
T5767	8951-8952	DT	denotes	a
T5769	8953-8956	NN	denotes	p53
T5771	8956-8957	HYPH	denotes	-
T5770	8957-8960	NN	denotes	GFP
T5772	8961-8967	NN	denotes	fusion
T5768	8968-8975	NN	denotes	protein
T5773	8975-8977	,	denotes	,
T5774	8977-8979	PRP	denotes	we
T5775	8980-8985	RB	denotes	first
T5766	8986-8995	VBD	denotes	subcloned
T5776	8996-8997	DT	denotes	a
T5778	8998-9003	NN	denotes	SacII
T5780	9003-9004	HYPH	denotes	-
T5779	9004-9011	NN	denotes	HindIII
T5777	9012-9020	NN	denotes	fragment
T5781	9021-9023	IN	denotes	of
T5782	9024-9027	DT	denotes	the
T5784	9028-9031	NN	denotes	p53
T5783	9032-9037	NN	denotes	locus
T5785	9038-9039	-LRB-	denotes	(
T5786	9039-9052	VBG	denotes	corresponding
T5787	9053-9055	IN	denotes	to
T5788	9056-9060	NN	denotes	part
T5789	9061-9063	IN	denotes	of
T5790	9064-9068	NN	denotes	exon
T5791	9069-9071	CD	denotes	10
T5792	9072-9074	IN	denotes	to
T5793	9075-9084	NNS	denotes	sequences
T5794	9085-9095	RB	denotes	downstream
T5795	9096-9098	IN	denotes	of
T5796	9099-9102	DT	denotes	the
T5797	9103-9107	NN	denotes	gene
T5798	9107-9108	-RRB-	denotes	)
T5799	9109-9113	IN	denotes	into
T5800	9114-9117	NN	denotes	pBS
T5801	9117-9119	,	denotes	,
T5802	9119-9123	RB	denotes	then
T5803	9124-9131	VBD	denotes	mutated
T5804	9132-9135	DT	denotes	the
T5806	9136-9143	NN	denotes	HindIII
T5805	9144-9148	NN	denotes	site
T5807	9149-9153	IN	denotes	into
T5808	9154-9155	DT	denotes	a
T5810	9156-9160	NN	denotes	FseI
T5809	9161-9165	NN	denotes	site
T5811	9165-9166	.	denotes	.
T5812	9166-9557	sentence	denotes	We next mutated the C-terminal part of the p53 gene in two rounds of PCR mutagenesis, first with primers 5′-GGGCCTGACTCAGACGGATCCCCTCTGCATCCCGTC-3′ and 5′-GACGGGATGCAGAGGGGATCCGTCTGAGTCAGGCCC-3′, which removed the stop codon and introduced a BamHI site, then with primers 5′-GACGGATCCCCTCTGAATTCCGTCCCCATCACCA-3′ and 5′-TGGTGATGGGGACGGAATACAGAGGGGATCCGTC-3′, which introduced an EcoRI site.
T5813	9167-9169	PRP	denotes	We
T5815	9170-9174	RB	denotes	next
T5814	9175-9182	VBD	denotes	mutated
T5816	9183-9186	DT	denotes	the
T5818	9187-9188	NN	denotes	C
T5820	9188-9189	HYPH	denotes	-
T5819	9189-9197	JJ	denotes	terminal
T5817	9198-9202	NN	denotes	part
T5821	9203-9205	IN	denotes	of
T5822	9206-9209	DT	denotes	the
T5824	9210-9213	NN	denotes	p53
T5823	9214-9218	NN	denotes	gene
T5825	9219-9221	IN	denotes	in
T5826	9222-9225	CD	denotes	two
T5827	9226-9232	NNS	denotes	rounds
T5828	9233-9235	IN	denotes	of
T5829	9236-9239	NN	denotes	PCR
T5830	9240-9251	NN	denotes	mutagenesis
T5831	9251-9253	,	denotes	,
T5832	9253-9258	RB	denotes	first
T5833	9259-9263	IN	denotes	with
T5834	9264-9271	NNS	denotes	primers
T5836	9272-9273	CD	denotes	5
T5837	9273-9274	SYM	denotes	′
T5838	9274-9275	HYPH	denotes	-
T5835	9275-9311	NN	denotes	GGGCCTGACTCAGACGGATCCCCTCTGCATCCCGTC
T5839	9311-9312	HYPH	denotes	-
T5840	9312-9313	CD	denotes	3
T5841	9313-9314	SYM	denotes	′
T5842	9315-9318	CC	denotes	and
T5843	9319-9320	CD	denotes	5
T5845	9320-9321	SYM	denotes	′
T5846	9321-9322	HYPH	denotes	-
T5844	9322-9358	NN	denotes	GACGGGATGCAGAGGGGATCCGTCTGAGTCAGGCCC
T5847	9358-9359	HYPH	denotes	-
T5848	9359-9360	CD	denotes	3
T5849	9360-9361	SYM	denotes	′
T5850	9361-9363	,	denotes	,
T5851	9363-9368	WDT	denotes	which
T5852	9369-9376	VBD	denotes	removed
T5853	9377-9380	DT	denotes	the
T5855	9381-9385	NN	denotes	stop
T5854	9386-9391	NN	denotes	codon
T5856	9392-9395	CC	denotes	and
T5857	9396-9406	VBD	denotes	introduced
T5858	9407-9408	DT	denotes	a
T5860	9409-9414	NN	denotes	BamHI
T5859	9415-9419	NN	denotes	site
T5861	9419-9421	,	denotes	,
T5862	9421-9425	RB	denotes	then
T5863	9426-9430	IN	denotes	with
T5864	9431-9438	NNS	denotes	primers
T5866	9439-9440	CD	denotes	5
T5867	9440-9441	SYM	denotes	′
T5868	9441-9442	HYPH	denotes	-
T5865	9442-9476	NN	denotes	GACGGATCCCCTCTGAATTCCGTCCCCATCACCA
T5869	9476-9477	HYPH	denotes	-
T5870	9477-9478	CD	denotes	3
T5871	9478-9479	SYM	denotes	′
T5872	9480-9483	CC	denotes	and
T5873	9484-9485	CD	denotes	5
T5875	9485-9486	SYM	denotes	′
T5876	9486-9487	HYPH	denotes	-
T5874	9487-9521	NN	denotes	TGGTGATGGGGACGGAATACAGAGGGGATCCGTC
T5877	9521-9522	HYPH	denotes	-
T5878	9522-9523	CD	denotes	3
T5879	9523-9524	SYM	denotes	′
T5880	9524-9526	,	denotes	,
T5881	9526-9531	WDT	denotes	which
T5882	9532-9542	VBD	denotes	introduced
T5883	9543-9545	DT	denotes	an
T5885	9546-9551	NN	denotes	EcoRI
T5884	9552-9556	NN	denotes	site
T5886	9556-9557	.	denotes	.
T5887	9557-9744	sentence	denotes	We verified the sequence from the mutated plasmid, then digested it with BamHI and EcoRI, to insert in frame GFP sequences from a Bam HI-EcoRI fragment of plasmid phr-GFP-1 (Stratagene).
T5888	9558-9560	PRP	denotes	We
T5889	9561-9569	VBD	denotes	verified
T5890	9570-9573	DT	denotes	the
T5891	9574-9582	NN	denotes	sequence
T5892	9583-9587	IN	denotes	from
T5893	9588-9591	DT	denotes	the
T5895	9592-9599	VBN	denotes	mutated
T5894	9600-9607	NN	denotes	plasmid
T5896	9607-9609	,	denotes	,
T5897	9609-9613	RB	denotes	then
T5898	9614-9622	VBD	denotes	digested
T5899	9623-9625	PRP	denotes	it
T5900	9626-9630	IN	denotes	with
T5901	9631-9636	NN	denotes	BamHI
T5902	9637-9640	CC	denotes	and
T5903	9641-9646	NN	denotes	EcoRI
T5904	9646-9648	,	denotes	,
T5905	9648-9650	TO	denotes	to
T5906	9651-9657	VB	denotes	insert
T5907	9658-9660	IN	denotes	in
T5908	9661-9666	NN	denotes	frame
T5909	9667-9670	NN	denotes	GFP
T5910	9671-9680	NNS	denotes	sequences
T5911	9681-9685	IN	denotes	from
T5912	9686-9687	DT	denotes	a
T5914	9688-9691	NN	denotes	Bam
T5916	9692-9694	NN	denotes	HI
T5917	9694-9695	HYPH	denotes	-
T5915	9695-9700	NN	denotes	EcoRI
T5913	9701-9709	NN	denotes	fragment
T5918	9710-9712	IN	denotes	of
T5919	9713-9720	NN	denotes	plasmid
T5921	9721-9724	NN	denotes	phr
T5922	9724-9725	HYPH	denotes	-
T5920	9725-9728	NN	denotes	GFP
T5923	9728-9729	HYPH	denotes	-
T5924	9729-9730	CD	denotes	1
T5925	9731-9732	-LRB-	denotes	(
T5926	9732-9742	NNP	denotes	Stratagene
T5927	9742-9743	-RRB-	denotes	)
T5928	9743-9744	.	denotes	.
T5929	9744-9987	sentence	denotes	We verified the sequence of this p53-GFP fusion fragment, then swapped it in the L3-p53PmlEagPuroΔTK-1L plasmid (see above) by HindIII and FseI digestion, resulting in the p53GFP exchange construct, the sequence which was verified before use.
T5930	9745-9747	PRP	denotes	We
T5931	9748-9756	VBD	denotes	verified
T5932	9757-9760	DT	denotes	the
T5933	9761-9769	NN	denotes	sequence
T5934	9770-9772	IN	denotes	of
T5935	9773-9777	DT	denotes	this
T5937	9778-9781	NN	denotes	p53
T5939	9781-9782	HYPH	denotes	-
T5938	9782-9785	NN	denotes	GFP
T5940	9786-9792	NN	denotes	fusion
T5936	9793-9801	NN	denotes	fragment
T5941	9801-9803	,	denotes	,
T5942	9803-9807	RB	denotes	then
T5943	9808-9815	VBD	denotes	swapped
T5944	9816-9818	PRP	denotes	it
T5945	9819-9821	IN	denotes	in
T5946	9822-9825	DT	denotes	the
T5948	9826-9828	NN	denotes	L3
T5950	9828-9829	HYPH	denotes	-
T5951	9829-9845	NN	denotes	p53PmlEagPuroΔTK
T5952	9845-9846	HYPH	denotes	-
T5949	9846-9848	NN	denotes	1L
T5947	9849-9856	NN	denotes	plasmid
T5953	9857-9858	-LRB-	denotes	(
T5954	9858-9861	VB	denotes	see
T5955	9862-9867	RB	denotes	above
T5956	9867-9868	-RRB-	denotes	)
T5957	9869-9871	IN	denotes	by
T5958	9872-9879	NN	denotes	HindIII
T5960	9880-9883	CC	denotes	and
T5961	9884-9888	NN	denotes	FseI
T5959	9889-9898	NN	denotes	digestion
T5962	9898-9900	,	denotes	,
T5963	9900-9909	VBG	denotes	resulting
T5964	9910-9912	IN	denotes	in
T5965	9913-9916	DT	denotes	the
T5967	9917-9923	NN	denotes	p53GFP
T5968	9924-9932	NN	denotes	exchange
T5966	9933-9942	NN	denotes	construct
T5969	9942-9944	,	denotes	,
T5970	9944-9947	DT	denotes	the
T5971	9948-9956	NN	denotes	sequence
T5972	9957-9962	WDT	denotes	which
T5974	9963-9966	VBD	denotes	was
T5973	9967-9975	VBN	denotes	verified
T5975	9976-9982	IN	denotes	before
T5976	9983-9986	NN	denotes	use
T5977	9986-9987	.	denotes	.
T5978	9987-10163	sentence	denotes	The p53ΔPGFP exchange construct was engineered by combining sequences from the p53GFP exchange plasmid and sequences from the p53ΔP targeting construct described recently (5).
T5979	9988-9991	DT	denotes	The
T5981	9992-10000	NN	denotes	p53ΔPGFP
T5982	10001-10009	NN	denotes	exchange
T5980	10010-10019	NN	denotes	construct
T5984	10020-10023	VBD	denotes	was
T5983	10024-10034	VBN	denotes	engineered
T5985	10035-10037	IN	denotes	by
T5986	10038-10047	VBG	denotes	combining
T5987	10048-10057	NNS	denotes	sequences
T5988	10058-10062	IN	denotes	from
T5989	10063-10066	DT	denotes	the
T5991	10067-10073	NN	denotes	p53GFP
T5992	10074-10082	NN	denotes	exchange
T5990	10083-10090	NN	denotes	plasmid
T5993	10091-10094	CC	denotes	and
T5994	10095-10104	NNS	denotes	sequences
T5995	10105-10109	IN	denotes	from
T5996	10110-10113	DT	denotes	the
T5998	10114-10119	NN	denotes	p53ΔP
T5999	10120-10129	NN	denotes	targeting
T5997	10130-10139	NN	denotes	construct
T6000	10140-10149	VBN	denotes	described
T6001	10150-10158	RB	denotes	recently
T6002	10159-10160	-LRB-	denotes	(
T6003	10160-10161	CD	denotes	5
T6004	10161-10162	-RRB-	denotes	)
T6005	10162-10163	.	denotes	.
T6006	10163-10206	sentence	denotes	Its sequence was also verified before use.
T6007	10164-10167	PRP$	denotes	Its
T6008	10168-10176	NN	denotes	sequence
T6010	10177-10180	VBD	denotes	was
T6011	10181-10185	RB	denotes	also
T6009	10186-10194	VBN	denotes	verified
T6012	10195-10201	IN	denotes	before
T6013	10202-10205	NN	denotes	use
T6014	10205-10206	.	denotes	.
T6352	10208-10217	NNS	denotes	Sequences
T6353	10218-10221	CC	denotes	and
T6354	10222-10225	NN	denotes	use
T6355	10226-10228	IN	denotes	of
T6356	10229-10232	NN	denotes	PCR
T6357	10233-10240	NNS	denotes	primers
T6358	10240-11040	sentence	denotes	a: 5′-CCCCGGCCCTCACCCTCATCTTCG-3′, from the PuΔTK gene, assays targeting of Flox plasmid; b: 5′-AACAAAACAAAACAGCAGCAACAA-3′, from sequences downstream of the p53 gene and outside Flox sequences, assays targeting of Flox and RMCE with p53GFP or p53ΔPGFP plasmids; c: 5′-TGAAGAGCAAGGGCGTGGTGAAGGA-3′, from GFP sequences, assays RMCE with p53GFP orp53ΔPGFP plasmids; d: 5′-CAAAAAATGGAAGGAAATCAGGAACTAA-3′, from p53 intron 3, and e: 5′-TCTAGACAGAGAAAAAAGAGGCATT-3′, from p53 intron 4, assay RMCE with p53ΔPGFP plasmid; f: 5′-ATGGGAGGCTGCCAGTCCTAACCC and g: 5′-GTGTTTCATTAGTTCCCCACCTTGAC-3′ amplify the WT p53 allele according to Taconic's procedures, h: 5′-TTTACGGAGCCCTGGCGCTCGATGT-3′ and i: 5′-GTGGGAGGGACAAAAGTTCGAGGCC-3′ amplify the Neo marker in the p53 KO allele according to Taconic's procedures.
T6359	10241-10242	LS	denotes	a
T6361	10242-10244	:	denotes	:
T6362	10244-10245	CD	denotes	5
T6363	10245-10246	SYM	denotes	′
T6364	10246-10247	HYPH	denotes	-
T6360	10247-10271	NN	denotes	CCCCGGCCCTCACCCTCATCTTCG
T6366	10271-10272	HYPH	denotes	-
T6367	10272-10273	CD	denotes	3
T6368	10273-10274	SYM	denotes	′
T6369	10274-10276	,	denotes	,
T6370	10276-10280	IN	denotes	from
T6371	10281-10284	DT	denotes	the
T6373	10285-10290	NN	denotes	PuΔTK
T6372	10291-10295	NN	denotes	gene
T6374	10295-10297	,	denotes	,
T6375	10297-10303	NNS	denotes	assays
T6376	10304-10313	VBG	denotes	targeting
T6377	10314-10316	IN	denotes	of
T6378	10317-10321	NN	denotes	Flox
T6379	10322-10329	NN	denotes	plasmid
T6380	10329-10330	:	denotes	;
T6381	10331-10332	LS	denotes	b
T6383	10332-10334	:	denotes	:
T6384	10334-10335	CD	denotes	5
T6385	10335-10336	SYM	denotes	′
T6386	10336-10337	HYPH	denotes	-
T6382	10337-10361	NN	denotes	AACAAAACAAAACAGCAGCAACAA
T6387	10361-10362	HYPH	denotes	-
T6388	10362-10363	CD	denotes	3
T6389	10363-10364	SYM	denotes	′
T6390	10364-10366	,	denotes	,
T6391	10366-10370	IN	denotes	from
T6392	10371-10380	NNS	denotes	sequences
T6393	10381-10391	RB	denotes	downstream
T6395	10392-10394	IN	denotes	of
T6396	10395-10398	DT	denotes	the
T6398	10399-10402	NN	denotes	p53
T6397	10403-10407	NN	denotes	gene
T6399	10408-10411	CC	denotes	and
T6394	10412-10419	IN	denotes	outside
T6400	10420-10424	NN	denotes	Flox
T6401	10425-10434	NNS	denotes	sequences
T6402	10434-10436	,	denotes	,
T6403	10436-10442	NNS	denotes	assays
T6404	10443-10452	VBG	denotes	targeting
T6405	10453-10455	IN	denotes	of
T6406	10456-10460	NN	denotes	Flox
T6407	10461-10464	CC	denotes	and
T6408	10465-10469	NN	denotes	RMCE
T6409	10470-10474	IN	denotes	with
T6410	10475-10481	NN	denotes	p53GFP
T6412	10482-10484	CC	denotes	or
T6413	10485-10493	NN	denotes	p53ΔPGFP
T6411	10494-10502	NNS	denotes	plasmids
T6414	10502-10503	:	denotes	;
T6415	10504-10505	LS	denotes	c
T6417	10505-10507	:	denotes	:
T6418	10507-10508	CD	denotes	5
T6419	10508-10509	SYM	denotes	′
T6420	10509-10510	HYPH	denotes	-
T6416	10510-10535	NN	denotes	TGAAGAGCAAGGGCGTGGTGAAGGA
T6421	10535-10536	HYPH	denotes	-
T6422	10536-10537	CD	denotes	3
T6423	10537-10538	SYM	denotes	′
T6424	10538-10540	,	denotes	,
T6425	10540-10544	IN	denotes	from
T6426	10545-10548	NN	denotes	GFP
T6427	10549-10558	NNS	denotes	sequences
T6428	10558-10560	,	denotes	,
T6429	10560-10566	NNS	denotes	assays
T6430	10567-10571	NN	denotes	RMCE
T6431	10572-10576	IN	denotes	with
T6432	10577-10583	NN	denotes	p53GFP
T6433	10584-10594	NN	denotes	orp53ΔPGFP
T6434	10595-10603	NNS	denotes	plasmids
T6435	10603-10604	:	denotes	;
T6436	10605-10606	LS	denotes	d
T6438	10606-10608	:	denotes	:
T6439	10608-10609	CD	denotes	5
T6440	10609-10610	SYM	denotes	′
T6441	10610-10611	HYPH	denotes	-
T6437	10611-10639	NN	denotes	CAAAAAATGGAAGGAAATCAGGAACTAA
T6442	10639-10640	HYPH	denotes	-
T6443	10640-10641	CD	denotes	3
T6444	10641-10642	SYM	denotes	′
T6445	10642-10644	,	denotes	,
T6446	10644-10648	IN	denotes	from
T6447	10649-10652	NN	denotes	p53
T6448	10653-10659	NN	denotes	intron
T6449	10660-10661	CD	denotes	3
T6450	10661-10663	,	denotes	,
T6451	10663-10666	CC	denotes	and
T6452	10667-10668	LS	denotes	e
T6454	10668-10670	:	denotes	:
T6455	10670-10671	CD	denotes	5
T6456	10671-10672	SYM	denotes	′
T6457	10672-10673	HYPH	denotes	-
T6453	10673-10698	NN	denotes	TCTAGACAGAGAAAAAAGAGGCATT
T6458	10698-10699	HYPH	denotes	-
T6459	10699-10700	CD	denotes	3
T6460	10700-10701	SYM	denotes	′
T6461	10701-10703	,	denotes	,
T6462	10703-10707	IN	denotes	from
T6463	10708-10711	NN	denotes	p53
T6464	10712-10718	NN	denotes	intron
T6465	10719-10720	CD	denotes	4
T6466	10720-10722	,	denotes	,
T6467	10722-10727	NN	denotes	assay
T6468	10728-10732	NN	denotes	RMCE
T6469	10733-10737	IN	denotes	with
T6470	10738-10746	NN	denotes	p53ΔPGFP
T6471	10747-10754	NN	denotes	plasmid
T6472	10754-10755	:	denotes	;
T6473	10756-10757	LS	denotes	f
T6475	10757-10759	:	denotes	:
T6476	10759-10760	CD	denotes	5
T6477	10760-10761	SYM	denotes	′
T6478	10761-10762	HYPH	denotes	-
T6474	10762-10786	NN	denotes	ATGGGAGGCTGCCAGTCCTAACCC
T6479	10787-10790	CC	denotes	and
T6480	10791-10792	LS	denotes	g
T6482	10792-10794	:	denotes	:
T6483	10794-10795	CD	denotes	5
T6484	10795-10796	SYM	denotes	′
T6485	10796-10797	HYPH	denotes	-
T6481	10797-10823	NN	denotes	GTGTTTCATTAGTTCCCCACCTTGAC
T6486	10823-10824	HYPH	denotes	-
T6487	10824-10825	CD	denotes	3
T6488	10825-10826	SYM	denotes	′
T6365	10827-10834	VBP	denotes	amplify
T6490	10835-10838	DT	denotes	the
T6492	10839-10841	NN	denotes	WT
T6493	10842-10845	NN	denotes	p53
T6491	10846-10852	NN	denotes	allele
T6494	10853-10862	IN	denotes	according
T6495	10863-10865	IN	denotes	to
T6496	10866-10873	NNP	denotes	Taconic
T6498	10873-10875	POS	denotes	's
T6497	10876-10886	NNS	denotes	procedures
T6499	10886-10888	,	denotes	,
T6500	10888-10889	LS	denotes	h
T6502	10889-10891	:	denotes	:
T6503	10891-10892	CD	denotes	5
T6504	10892-10893	SYM	denotes	′
T6505	10893-10894	HYPH	denotes	-
T6501	10894-10919	NN	denotes	TTTACGGAGCCCTGGCGCTCGATGT
T6506	10919-10920	HYPH	denotes	-
T6507	10920-10921	CD	denotes	3
T6508	10921-10922	SYM	denotes	′
T6509	10923-10926	CC	denotes	and
T6510	10927-10928	LS	denotes	i
T6512	10928-10930	:	denotes	:
T6513	10930-10931	CD	denotes	5
T6514	10931-10932	SYM	denotes	′
T6515	10932-10933	HYPH	denotes	-
T6511	10933-10958	NN	denotes	GTGGGAGGGACAAAAGTTCGAGGCC
T6516	10958-10959	HYPH	denotes	-
T6517	10959-10960	CD	denotes	3
T6518	10960-10961	SYM	denotes	′
T6489	10962-10969	VBP	denotes	amplify
T6519	10970-10973	DT	denotes	the
T6521	10974-10977	NN	denotes	Neo
T6520	10978-10984	NN	denotes	marker
T6522	10985-10987	IN	denotes	in
T6523	10988-10991	DT	denotes	the
T6525	10992-10995	NN	denotes	p53
T6526	10996-10998	NN	denotes	KO
T6524	10999-11005	NN	denotes	allele
T6527	11006-11015	IN	denotes	according
T6528	11016-11018	IN	denotes	to
T6529	11019-11026	NNP	denotes	Taconic
T6531	11026-11028	POS	denotes	's
T6530	11029-11039	NNS	denotes	procedures
T6532	11039-11040	.	denotes	.
T6727	11042-11046	NN	denotes	Cell
T6728	11047-11054	NN	denotes	culture
T6729	11055-11065	NNS	denotes	conditions
T6730	11065-11193	sentence	denotes	Primary MEFs, isolated from 13.5 day embryos, were cultured in DMEM with 15% FBS, 100 mM BME, 2 mM l-glutamine and antibiotics.
T6731	11066-11073	JJ	denotes	Primary
T6732	11074-11078	NNS	denotes	MEFs
T6734	11078-11080	,	denotes	,
T6735	11080-11088	VBN	denotes	isolated
T6736	11089-11093	IN	denotes	from
T6737	11094-11098	CD	denotes	13.5
T6738	11099-11102	NN	denotes	day
T6739	11103-11110	NNS	denotes	embryos
T6740	11110-11112	,	denotes	,
T6741	11112-11116	VBD	denotes	were
T6733	11117-11125	VBN	denotes	cultured
T6742	11126-11128	IN	denotes	in
T6743	11129-11133	NN	denotes	DMEM
T6744	11134-11138	IN	denotes	with
T6745	11139-11141	CD	denotes	15
T6746	11141-11142	NN	denotes	%
T6747	11143-11146	NN	denotes	FBS
T6748	11146-11148	,	denotes	,
T6749	11148-11151	CD	denotes	100
T6750	11152-11154	NN	denotes	mM
T6751	11155-11158	NN	denotes	BME
T6752	11158-11160	,	denotes	,
T6753	11160-11161	CD	denotes	2
T6754	11162-11164	NN	denotes	mM
T6756	11165-11166	NN	denotes	l
T6757	11166-11167	HYPH	denotes	-
T6755	11167-11176	NN	denotes	glutamine
T6758	11177-11180	CC	denotes	and
T6759	11181-11192	NNS	denotes	antibiotics
T6760	11192-11193	.	denotes	.
T6761	11193-11369	sentence	denotes	129/SvJae ES cells were grown in the same medium supplemented with 1000 U/ml ESGRO (Chemicon), on a layer of mitomycin C-treated SNLPuro-7/4 feeders (kind gift of A. Bradley).
T6762	11194-11197	CD	denotes	129
T6764	11197-11198	HYPH	denotes	/
T6763	11198-11203	NN	denotes	SvJae
T6766	11204-11206	NN	denotes	ES
T6765	11207-11212	NNS	denotes	cells
T6768	11213-11217	VBD	denotes	were
T6767	11218-11223	VBN	denotes	grown
T6769	11224-11226	IN	denotes	in
T6770	11227-11230	DT	denotes	the
T6772	11231-11235	JJ	denotes	same
T6771	11236-11242	NN	denotes	medium
T6773	11243-11255	VBN	denotes	supplemented
T6774	11256-11260	IN	denotes	with
T6775	11261-11265	CD	denotes	1000
T6776	11266-11267	NN	denotes	U
T6778	11267-11268	SYM	denotes	/
T6779	11268-11270	NN	denotes	ml
T6777	11271-11276	NN	denotes	ESGRO
T6780	11277-11278	-LRB-	denotes	(
T6781	11278-11286	NNP	denotes	Chemicon
T6782	11286-11287	-RRB-	denotes	)
T6783	11287-11289	,	denotes	,
T6784	11289-11291	IN	denotes	on
T6785	11292-11293	DT	denotes	a
T6786	11294-11299	NN	denotes	layer
T6787	11300-11302	IN	denotes	of
T6788	11303-11312	NN	denotes	mitomycin
T6789	11313-11314	NN	denotes	C
T6791	11314-11315	HYPH	denotes	-
T6790	11315-11322	JJ	denotes	treated
T6793	11323-11330	NN	denotes	SNLPuro
T6794	11330-11331	HYPH	denotes	-
T6795	11331-11332	CD	denotes	7
T6796	11332-11333	HYPH	denotes	/
T6797	11333-11334	CD	denotes	4
T6792	11335-11342	NNS	denotes	feeders
T6798	11343-11344	-LRB-	denotes	(
T6800	11344-11348	JJ	denotes	kind
T6799	11349-11353	NN	denotes	gift
T6801	11354-11356	IN	denotes	of
T6802	11357-11359	NNP	denotes	A.
T6803	11360-11367	NNP	denotes	Bradley
T6804	11367-11368	-RRB-	denotes	)
T6805	11368-11369	.	denotes	.
T6806	11369-11452	sentence	denotes	Selections were performed with 2 μg/ml puromycin, 0.2 μM FIAU or 2 μM ganciclovir.
T6807	11370-11380	NNS	denotes	Selections
T6809	11381-11385	VBD	denotes	were
T6808	11386-11395	VBN	denotes	performed
T6810	11396-11400	IN	denotes	with
T6811	11401-11402	CD	denotes	2
T6812	11403-11405	NN	denotes	μg
T6814	11405-11406	SYM	denotes	/
T6815	11406-11408	NN	denotes	ml
T6813	11409-11418	NN	denotes	puromycin
T6816	11418-11420	,	denotes	,
T6817	11420-11423	CD	denotes	0.2
T6818	11424-11426	NN	denotes	μM
T6819	11427-11431	NN	denotes	FIAU
T6820	11432-11434	CC	denotes	or
T6821	11435-11436	CD	denotes	2
T6822	11437-11439	NN	denotes	μM
T6823	11440-11451	NN	denotes	ganciclovir
T6824	11451-11452	.	denotes	.
T6929	11454-11463	NN	denotes	Targeting
T6931	11463-11464	HYPH	denotes	/
T6930	11464-11474	NN	denotes	genotyping
T6932	11475-11477	IN	denotes	of
T6933	11478-11481	DT	denotes	the
T6935	11482-11486	JJ	denotes	RMCE
T6937	11486-11487	HYPH	denotes	-
T6936	11487-11492	JJ	denotes	ready
T6934	11493-11498	NN	denotes	locus
T6938	11498-11652	sentence	denotes	29/SvJae ES cells were electroporated with the Flox construct linearized with PmeI, and puromycin resistant clones were analyzed as described (Figure 2).
T6939	11499-11501	CD	denotes	29
T6941	11501-11502	HYPH	denotes	/
T6940	11502-11507	NN	denotes	SvJae
T6943	11508-11510	NN	denotes	ES
T6942	11511-11516	NNS	denotes	cells
T6945	11517-11521	VBD	denotes	were
T6944	11522-11536	VBN	denotes	electroporated
T6946	11537-11541	IN	denotes	with
T6947	11542-11545	DT	denotes	the
T6949	11546-11550	NN	denotes	Flox
T6948	11551-11560	NN	denotes	construct
T6950	11561-11571	VBN	denotes	linearized
T6951	11572-11576	IN	denotes	with
T6952	11577-11581	NN	denotes	PmeI
T6953	11581-11583	,	denotes	,
T6954	11583-11586	CC	denotes	and
T6955	11587-11596	NN	denotes	puromycin
T6956	11597-11606	JJ	denotes	resistant
T6957	11607-11613	NNS	denotes	clones
T6959	11614-11618	VBD	denotes	were
T6958	11619-11627	VBN	denotes	analyzed
T6960	11628-11630	IN	denotes	as
T6961	11631-11640	VBN	denotes	described
T6962	11641-11642	-LRB-	denotes	(
T6963	11642-11648	NN	denotes	Figure
T6964	11649-11650	CD	denotes	2
T6965	11650-11651	-RRB-	denotes	)
T6966	11651-11652	.	denotes	.
T6967	11652-11732	sentence	denotes	Two clones were injected into blastocysts and transmitted through the germline.
T6968	11653-11656	CD	denotes	Two
T6969	11657-11663	NNS	denotes	clones
T6971	11664-11668	VBD	denotes	were
T6970	11669-11677	VBN	denotes	injected
T6972	11678-11682	IN	denotes	into
T6973	11683-11694	NNS	denotes	blastocysts
T6974	11695-11698	CC	denotes	and
T6975	11699-11710	VBN	denotes	transmitted
T6976	11711-11718	IN	denotes	through
T6977	11719-11722	DT	denotes	the
T6978	11723-11731	NN	denotes	germline
T6979	11731-11732	.	denotes	.
T7192	11734-11744	VBG	denotes	Performing
T7193	11745-11749	NN	denotes	RMCE
T7194	11750-11752	IN	denotes	in
T7195	11753-11755	NN	denotes	ES
T7196	11756-11761	NNS	denotes	cells
T7197	11761-11976	sentence	denotes	A total of 8 × 105 p53RMCE/+ ES cells were grown without puromycin for 12 h, electroporated with 15 μg CMV-Cre plasmid (pOG231) and 200 μg of the exchange construct, and plated in T25 flasks at 105 cells per flask.
T7198	11762-11763	DT	denotes	A
T7199	11764-11769	NN	denotes	total
T7201	11770-11772	IN	denotes	of
T7202	11773-11774	CD	denotes	8
T7204	11775-11776	HYPH	denotes	×
T7203	11777-11780	CD	denotes	105
T7206	11781-11788	NN	denotes	p53RMCE
T7207	11788-11789	HYPH	denotes	/
T7208	11789-11790	SYM	denotes	+
T7209	11791-11793	NN	denotes	ES
T7205	11794-11799	NNS	denotes	cells
T7210	11800-11804	VBD	denotes	were
T7200	11805-11810	VBN	denotes	grown
T7211	11811-11818	IN	denotes	without
T7212	11819-11828	NN	denotes	puromycin
T7213	11829-11832	IN	denotes	for
T7214	11833-11835	CD	denotes	12
T7215	11836-11837	NN	denotes	h
T7216	11837-11839	,	denotes	,
T7217	11839-11853	VBN	denotes	electroporated
T7218	11854-11858	IN	denotes	with
T7219	11859-11861	CD	denotes	15
T7221	11862-11864	NN	denotes	μg
T7222	11865-11868	NN	denotes	CMV
T7224	11868-11869	HYPH	denotes	-
T7223	11869-11872	NN	denotes	Cre
T7220	11873-11880	NN	denotes	plasmid
T7225	11881-11882	-LRB-	denotes	(
T7226	11882-11888	NN	denotes	pOG231
T7227	11888-11889	-RRB-	denotes	)
T7228	11890-11893	CC	denotes	and
T7229	11894-11897	CD	denotes	200
T7230	11898-11900	NN	denotes	μg
T7231	11901-11903	IN	denotes	of
T7232	11904-11907	DT	denotes	the
T7234	11908-11916	NN	denotes	exchange
T7233	11917-11926	NN	denotes	construct
T7235	11926-11928	,	denotes	,
T7236	11928-11931	CC	denotes	and
T7237	11932-11938	VBN	denotes	plated
T7238	11939-11941	IN	denotes	in
T7239	11942-11945	NN	denotes	T25
T7240	11946-11952	NNS	denotes	flasks
T7241	11953-11955	IN	denotes	at
T7242	11956-11959	CD	denotes	105
T7243	11960-11965	NNS	denotes	cells
T7244	11966-11969	IN	denotes	per
T7245	11970-11975	NN	denotes	flask
T7246	11975-11976	.	denotes	.
T7247	11976-12037	sentence	denotes	FIAU was added to the medium 3–4 days after electroporation.
T7248	11977-11981	NN	denotes	FIAU
T7250	11982-11985	VBD	denotes	was
T7249	11986-11991	VBN	denotes	added
T7251	11992-11994	IN	denotes	to
T7252	11995-11998	DT	denotes	the
T7253	11999-12005	NN	denotes	medium
T7254	12006-12007	CD	denotes	3
T7256	12007-12008	HYPH	denotes	–
T7255	12008-12009	CD	denotes	4
T7257	12010-12014	NNS	denotes	days
T7258	12015-12020	IN	denotes	after
T7259	12021-12036	NN	denotes	electroporation
T7260	12036-12037	.	denotes	.
T7261	12037-12216	sentence	denotes	Individual clones, picked 10 days after electroporation, were grown in 96-well plates and expanded to generate duplicate plates for freezing and DNA analysis by PCR and Southern.
T7262	12038-12048	JJ	denotes	Individual
T7263	12049-12055	NNS	denotes	clones
T7265	12055-12057	,	denotes	,
T7266	12057-12063	VBN	denotes	picked
T7267	12064-12066	CD	denotes	10
T7268	12067-12071	NNS	denotes	days
T7269	12072-12077	IN	denotes	after
T7270	12078-12093	NN	denotes	electroporation
T7271	12093-12095	,	denotes	,
T7272	12095-12099	VBD	denotes	were
T7264	12100-12105	VBN	denotes	grown
T7273	12106-12108	IN	denotes	in
T7274	12109-12111	CD	denotes	96
T7276	12111-12112	HYPH	denotes	-
T7275	12112-12116	NN	denotes	well
T7277	12117-12123	NNS	denotes	plates
T7278	12124-12127	CC	denotes	and
T7279	12128-12136	VBN	denotes	expanded
T7280	12137-12139	TO	denotes	to
T7281	12140-12148	VB	denotes	generate
T7282	12149-12158	JJ	denotes	duplicate
T7283	12159-12165	NNS	denotes	plates
T7284	12166-12169	IN	denotes	for
T7285	12170-12178	VBG	denotes	freezing
T7286	12179-12182	CC	denotes	and
T7287	12183-12186	NN	denotes	DNA
T7288	12187-12195	NN	denotes	analysis
T7289	12196-12198	IN	denotes	by
T7290	12199-12202	NN	denotes	PCR
T7291	12203-12206	CC	denotes	and
T7292	12207-12215	NNP	denotes	Southern
T7293	12215-12216	.	denotes	.
T7497	12218-12228	VBG	denotes	Performing
T7498	12229-12233	NN	denotes	RMCE
T7499	12234-12236	IN	denotes	in
T7500	12237-12241	NNS	denotes	MEFs
T7501	12241-12482	sentence	denotes	A total of 106 p53RMCE/− MEFs cells were grown without puromycin for 12 h, electroporated with 3 μg pOG231 and 30 μg exchange construct, and plated in a single 10 cm-dish, grown for 3 days then split in several dishes at 105 cells per dish.
T7502	12242-12243	DT	denotes	A
T7503	12244-12249	NN	denotes	total
T7505	12250-12252	IN	denotes	of
T7506	12253-12256	CD	denotes	106
T7508	12257-12264	NN	denotes	p53RMCE
T7510	12264-12265	HYPH	denotes	/
T7511	12265-12266	SYM	denotes	−
T7509	12267-12271	NNS	denotes	MEFs
T7507	12272-12277	NNS	denotes	cells
T7512	12278-12282	VBD	denotes	were
T7504	12283-12288	VBN	denotes	grown
T7513	12289-12296	IN	denotes	without
T7514	12297-12306	NN	denotes	puromycin
T7515	12307-12310	IN	denotes	for
T7516	12311-12313	CD	denotes	12
T7517	12314-12315	NN	denotes	h
T7518	12315-12317	,	denotes	,
T7519	12317-12331	VBN	denotes	electroporated
T7520	12332-12336	IN	denotes	with
T7521	12337-12338	CD	denotes	3
T7522	12339-12341	NN	denotes	μg
T7523	12342-12348	NN	denotes	pOG231
T7524	12349-12352	CC	denotes	and
T7525	12353-12355	CD	denotes	30
T7526	12356-12358	NN	denotes	μg
T7528	12359-12367	NN	denotes	exchange
T7527	12368-12377	NN	denotes	construct
T7529	12377-12379	,	denotes	,
T7530	12379-12382	CC	denotes	and
T7531	12383-12389	VBN	denotes	plated
T7532	12390-12392	IN	denotes	in
T7533	12393-12394	DT	denotes	a
T7535	12395-12401	JJ	denotes	single
T7536	12402-12404	CD	denotes	10
T7537	12405-12407	NN	denotes	cm
T7538	12407-12408	HYPH	denotes	-
T7534	12408-12412	NN	denotes	dish
T7539	12412-12414	,	denotes	,
T7540	12414-12419	VBN	denotes	grown
T7541	12420-12423	IN	denotes	for
T7542	12424-12425	CD	denotes	3
T7543	12426-12430	NNS	denotes	days
T7544	12431-12435	RB	denotes	then
T7545	12436-12441	VBN	denotes	split
T7546	12442-12444	IN	denotes	in
T7547	12445-12452	JJ	denotes	several
T7548	12453-12459	NNS	denotes	dishes
T7549	12460-12462	IN	denotes	at
T7550	12463-12466	CD	denotes	105
T7551	12467-12472	NNS	denotes	cells
T7552	12473-12476	IN	denotes	per
T7553	12477-12481	NN	denotes	dish
T7554	12481-12482	.	denotes	.
T7555	12482-12570	sentence	denotes	FIAU or ganciclovir was added to the medium 4 days after electroporation, for 3–4 days.
T7556	12483-12487	NN	denotes	FIAU
T7558	12488-12490	CC	denotes	or
T7559	12491-12502	NN	denotes	ganciclovir
T7560	12503-12506	VBD	denotes	was
T7557	12507-12512	VBN	denotes	added
T7561	12513-12515	IN	denotes	to
T7562	12516-12519	DT	denotes	the
T7563	12520-12526	NN	denotes	medium
T7564	12527-12528	CD	denotes	4
T7565	12529-12533	NNS	denotes	days
T7566	12534-12539	IN	denotes	after
T7567	12540-12555	NN	denotes	electroporation
T7568	12555-12557	,	denotes	,
T7569	12557-12560	IN	denotes	for
T7570	12561-12562	CD	denotes	3
T7572	12562-12563	SYM	denotes	–
T7571	12563-12564	CD	denotes	4
T7573	12565-12569	NNS	denotes	days
T7574	12569-12570	.	denotes	.
T7575	12570-12689	sentence	denotes	Clones, picked 10 days after electroporation, were grown in 24-well plates and expanded for freezing and DNA analysis.
T7576	12571-12577	NNS	denotes	Clones
T7578	12577-12579	,	denotes	,
T7579	12579-12585	VBN	denotes	picked
T7580	12586-12588	CD	denotes	10
T7581	12589-12593	NNS	denotes	days
T7582	12594-12599	IN	denotes	after
T7583	12600-12615	NN	denotes	electroporation
T7584	12615-12617	,	denotes	,
T7585	12617-12621	VBD	denotes	were
T7577	12622-12627	VBN	denotes	grown
T7586	12628-12630	IN	denotes	in
T7587	12631-12633	CD	denotes	24
T7589	12633-12634	HYPH	denotes	-
T7588	12634-12638	NN	denotes	well
T7590	12639-12645	NNS	denotes	plates
T7591	12646-12649	CC	denotes	and
T7592	12650-12658	VBN	denotes	expanded
T7593	12659-12662	IN	denotes	for
T7594	12663-12671	NN	denotes	freezing
T7595	12672-12675	CC	denotes	and
T7596	12676-12679	NN	denotes	DNA
T7597	12680-12688	NN	denotes	analysis
T7598	12688-12689	.	denotes	.
T8070	12691-12698	NNP	denotes	Western
T8072	12698-12699	HYPH	denotes	-
T8071	12699-12704	NNS	denotes	blots
T8073	12704-13001	sentence	denotes	Cells, untreated or treated for 24 h with 0.5 μg/ml adriamycin, were lysed on the dish in a buffer consisting of 50 mM Tris (pH 8.0), 5 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM PMSF, 1 mM sodium vanadate, 10 mM NaF and Complete Mini Protease Inhibitors (Roche Diagnostics) at 4°C for 30 min.
T8074	12705-12710	NNS	denotes	Cells
T8076	12710-12712	,	denotes	,
T8077	12712-12721	JJ	denotes	untreated
T8078	12722-12724	CC	denotes	or
T8079	12725-12732	VBN	denotes	treated
T8080	12733-12736	IN	denotes	for
T8081	12737-12739	CD	denotes	24
T8082	12740-12741	NN	denotes	h
T8083	12742-12746	IN	denotes	with
T8084	12747-12750	CD	denotes	0.5
T8085	12751-12753	NN	denotes	μg
T8087	12753-12754	SYM	denotes	/
T8088	12754-12756	NN	denotes	ml
T8086	12757-12767	NN	denotes	adriamycin
T8089	12767-12769	,	denotes	,
T8090	12769-12773	VBD	denotes	were
T8075	12774-12779	VBN	denotes	lysed
T8091	12780-12782	IN	denotes	on
T8092	12783-12786	DT	denotes	the
T8093	12787-12791	NN	denotes	dish
T8094	12792-12794	IN	denotes	in
T8095	12795-12796	DT	denotes	a
T8096	12797-12803	NN	denotes	buffer
T8097	12804-12814	VBG	denotes	consisting
T8098	12815-12817	IN	denotes	of
T8099	12818-12820	CD	denotes	50
T8100	12821-12823	NN	denotes	mM
T8101	12824-12828	NN	denotes	Tris
T8102	12829-12830	-LRB-	denotes	(
T8103	12830-12832	NN	denotes	pH
T8104	12833-12836	CD	denotes	8.0
T8105	12836-12837	-RRB-	denotes	)
T8106	12837-12839	,	denotes	,
T8107	12839-12840	CD	denotes	5
T8108	12841-12843	NN	denotes	mM
T8109	12844-12848	NN	denotes	EDTA
T8110	12848-12850	,	denotes	,
T8111	12850-12853	CD	denotes	150
T8112	12854-12856	NN	denotes	mM
T8113	12857-12861	NN	denotes	NaCl
T8114	12861-12863	,	denotes	,
T8115	12863-12866	CD	denotes	0.5
T8116	12866-12867	NN	denotes	%
T8118	12868-12875	NN	denotes	Nonidet
T8119	12876-12877	NN	denotes	P
T8120	12877-12878	HYPH	denotes	-
T8117	12878-12880	NN	denotes	40
T8121	12880-12882	,	denotes	,
T8122	12882-12883	CD	denotes	1
T8123	12884-12886	NN	denotes	mM
T8124	12887-12891	NN	denotes	PMSF
T8125	12891-12893	,	denotes	,
T8126	12893-12894	CD	denotes	1
T8127	12895-12897	NN	denotes	mM
T8129	12898-12904	NN	denotes	sodium
T8128	12905-12913	NN	denotes	vanadate
T8130	12913-12915	,	denotes	,
T8131	12915-12917	CD	denotes	10
T8132	12918-12920	NN	denotes	mM
T8133	12921-12924	NNP	denotes	NaF
T8134	12925-12928	CC	denotes	and
T8135	12929-12937	NN	denotes	Complete
T8136	12938-12942	NN	denotes	Mini
T8138	12943-12951	NN	denotes	Protease
T8137	12952-12962	NNS	denotes	Inhibitors
T8139	12963-12964	-LRB-	denotes	(
T8141	12964-12969	NNP	denotes	Roche
T8140	12970-12981	NNP	denotes	Diagnostics
T8142	12981-12982	-RRB-	denotes	)
T8143	12983-12985	IN	denotes	at
T8144	12986-12987	CD	denotes	4
T8145	12987-12989	NNS	denotes	°C
T8146	12990-12993	IN	denotes	for
T8147	12994-12996	CD	denotes	30
T8148	12997-13000	NN	denotes	min
T8149	13000-13001	.	denotes	.
T8150	13001-13063	sentence	denotes	Lysates were scraped, then spun at 6000× g at 4°C for 10 min.
T8151	13002-13009	NNS	denotes	Lysates
T8153	13010-13014	VBD	denotes	were
T8152	13015-13022	VBN	denotes	scraped
T8154	13022-13024	,	denotes	,
T8155	13024-13028	RB	denotes	then
T8156	13029-13033	VBN	denotes	spun
T8157	13034-13036	IN	denotes	at
T8158	13037-13041	CD	denotes	6000
T8160	13041-13042	SYM	denotes	×
T8159	13043-13044	NN	denotes	g
T8161	13045-13047	IN	denotes	at
T8162	13048-13049	CD	denotes	4
T8163	13049-13051	NNS	denotes	°C
T8164	13052-13055	IN	denotes	for
T8165	13056-13058	CD	denotes	10
T8166	13059-13062	NN	denotes	min
T8167	13062-13063	.	denotes	.
T8168	13063-13155	sentence	denotes	Protein concentration in the supernatant was determined using the Bio-Rad DC protein assay.
T8169	13064-13071	NN	denotes	Protein
T8170	13072-13085	NN	denotes	concentration
T8172	13086-13088	IN	denotes	in
T8173	13089-13092	DT	denotes	the
T8174	13093-13104	NN	denotes	supernatant
T8175	13105-13108	VBD	denotes	was
T8171	13109-13119	VBN	denotes	determined
T8176	13120-13125	VBG	denotes	using
T8177	13126-13129	DT	denotes	the
T8179	13130-13133	NN	denotes	Bio
T8181	13133-13134	HYPH	denotes	-
T8180	13134-13137	NN	denotes	Rad
T8182	13138-13140	NN	denotes	DC
T8183	13141-13148	NN	denotes	protein
T8178	13149-13154	NN	denotes	assay
T8184	13154-13155	.	denotes	.
T8185	13155-13310	sentence	denotes	Lysates were separated on single percentage SDS/PAGE gels, then electrophoretically transferred to poly(vinylidene difluoride), using standard procedures.
T8186	13156-13163	NNS	denotes	Lysates
T8188	13164-13168	VBD	denotes	were
T8187	13169-13178	VBN	denotes	separated
T8189	13179-13181	IN	denotes	on
T8190	13182-13188	JJ	denotes	single
T8191	13189-13199	NN	denotes	percentage
T8193	13200-13203	NN	denotes	SDS
T8195	13203-13204	HYPH	denotes	/
T8194	13204-13208	NN	denotes	PAGE
T8192	13209-13213	NNS	denotes	gels
T8196	13213-13215	,	denotes	,
T8197	13215-13219	RB	denotes	then
T8199	13220-13239	RB	denotes	electrophoretically
T8198	13240-13251	VBN	denotes	transferred
T8200	13252-13254	IN	denotes	to
T8201	13255-13259	NN	denotes	poly
T8202	13259-13260	-LRB-	denotes	(
T8204	13260-13270	NN	denotes	vinylidene
T8203	13271-13281	NN	denotes	difluoride
T8205	13281-13282	-RRB-	denotes	)
T8206	13282-13284	,	denotes	,
T8207	13284-13289	VBG	denotes	using
T8208	13290-13298	JJ	denotes	standard
T8209	13299-13309	NNS	denotes	procedures
T8210	13309-13310	.	denotes	.
T8211	13310-13315	NNS	denotes	Blots
T8213	13310-13529	sentence	denotes	Blots were incubated in 5% non-fat dried milk in TBST (0.02 M Tris, pH 7.6/0.35 M NaCl/0.1% Tween-20) for 1 h at room temperature before probing with primary antibodies against p53 (CM-5, Novacastra) and -actin (Sigma).
T8214	13316-13320	VBD	denotes	were
T8212	13321-13330	VBN	denotes	incubated
T8215	13331-13333	IN	denotes	in
T8216	13334-13335	CD	denotes	5
T8217	13335-13336	NN	denotes	%
T8219	13337-13344	JJ	denotes	non-fat
T8220	13345-13350	VBN	denotes	dried
T8218	13351-13355	NN	denotes	milk
T8221	13356-13358	IN	denotes	in
T8222	13359-13363	NN	denotes	TBST
T8223	13364-13365	-LRB-	denotes	(
T8225	13365-13369	CD	denotes	0.02
T8226	13370-13371	NN	denotes	M
T8227	13372-13376	NN	denotes	Tris
T8228	13376-13378	,	denotes	,
T8229	13378-13380	NN	denotes	pH
T8230	13381-13384	CD	denotes	7.6
T8231	13384-13385	HYPH	denotes	/
T8232	13385-13389	CD	denotes	0.35
T8233	13390-13391	NN	denotes	M
T8234	13392-13396	NN	denotes	NaCl
T8235	13396-13397	HYPH	denotes	/
T8236	13397-13400	CD	denotes	0.1
T8237	13400-13401	NN	denotes	%
T8224	13402-13407	NN	denotes	Tween
T8238	13407-13408	HYPH	denotes	-
T8239	13408-13410	CD	denotes	20
T8240	13410-13411	-RRB-	denotes	)
T8241	13412-13415	IN	denotes	for
T8242	13416-13417	CD	denotes	1
T8243	13418-13419	NN	denotes	h
T8244	13420-13422	IN	denotes	at
T8245	13423-13427	NN	denotes	room
T8246	13428-13439	NN	denotes	temperature
T8247	13440-13446	IN	denotes	before
T8248	13447-13454	VBG	denotes	probing
T8249	13455-13459	IN	denotes	with
T8250	13460-13467	JJ	denotes	primary
T8251	13468-13478	NNS	denotes	antibodies
T8252	13479-13486	IN	denotes	against
T8253	13487-13490	NN	denotes	p53
T8254	13491-13492	-LRB-	denotes	(
T8256	13492-13494	NN	denotes	CM
T8257	13494-13495	HYPH	denotes	-
T8258	13495-13496	CD	denotes	5
T8259	13496-13498	,	denotes	,
T8255	13498-13508	NNP	denotes	Novacastra
T8260	13508-13509	-RRB-	denotes	)
T8261	13510-13513	CC	denotes	and
T8262	13514-13515	HYPH	denotes	-
T8263	13515-13520	NN	denotes	actin
T8264	13521-13522	-LRB-	denotes	(
T8265	13522-13527	NNP	denotes	Sigma
T8266	13527-13528	-RRB-	denotes	)
T8267	13528-13529	.	denotes	.
T8268	13529-13635	sentence	denotes	Secondary antibodies used include peroxidase-conjugated goat anti-mouse IgG and anti-rabbit IgG (Pierce).
T8269	13530-13539	JJ	denotes	Secondary
T8270	13540-13550	NNS	denotes	antibodies
T8272	13551-13555	VBN	denotes	used
T8271	13556-13563	VBP	denotes	include
T8273	13564-13574	NN	denotes	peroxidase
T8275	13574-13575	HYPH	denotes	-
T8274	13575-13585	VBN	denotes	conjugated
T8277	13586-13590	NN	denotes	goat
T8278	13591-13601	NN	denotes	anti-mouse
T8276	13602-13605	NN	denotes	IgG
T8279	13606-13609	CC	denotes	and
T8280	13610-13621	NN	denotes	anti-rabbit
T8281	13622-13625	NN	denotes	IgG
T8282	13626-13627	-LRB-	denotes	(
T8283	13627-13633	NNP	denotes	Pierce
T8284	13633-13634	-RRB-	denotes	)
T8285	13634-13635	.	denotes	.
T8286	13635-13752	sentence	denotes	Probed blots were incubated with Pierce Supersignal West Pico chemiluminescent substrate and exposed to X-ray films.
T8287	13636-13642	VBN	denotes	Probed
T8288	13643-13648	NNS	denotes	blots
T8290	13649-13653	VBD	denotes	were
T8289	13654-13663	VBN	denotes	incubated
T8291	13664-13668	IN	denotes	with
T8292	13669-13675	NNP	denotes	Pierce
T8294	13676-13687	NNP	denotes	Supersignal
T8295	13688-13692	NNP	denotes	West
T8296	13693-13697	NNP	denotes	Pico
T8297	13698-13714	JJ	denotes	chemiluminescent
T8293	13715-13724	NN	denotes	substrate
T8298	13725-13728	CC	denotes	and
T8299	13729-13736	VBN	denotes	exposed
T8300	13737-13739	IN	denotes	to
T8301	13740-13745	NN	denotes	X-ray
T8302	13746-13751	NNS	denotes	films
T8303	13751-13752	.	denotes	.
T8467	13754-13758	NN	denotes	Flow
T8468	13759-13768	NN	denotes	cytometry
T8469	13768-13879	sentence	denotes	Log phase cells were irradiated at RT with a 60 Co γ-irradiator at doses of 6 or 12 Gy and incubated for 24 h.
T8470	13769-13772	NN	denotes	Log
T8472	13773-13778	NN	denotes	phase
T8471	13779-13784	NNS	denotes	cells
T8474	13785-13789	VBD	denotes	were
T8473	13790-13800	VBN	denotes	irradiated
T8475	13801-13803	IN	denotes	at
T8476	13804-13806	NN	denotes	RT
T8477	13807-13811	IN	denotes	with
T8478	13812-13813	DT	denotes	a
T8480	13814-13816	CD	denotes	60
T8482	13817-13819	NN	denotes	Co
T8481	13820-13821	NN	denotes	γ
T8483	13821-13822	SYM	denotes	-
T8479	13822-13832	NN	denotes	irradiator
T8484	13833-13835	IN	denotes	at
T8485	13836-13841	NNS	denotes	doses
T8486	13842-13844	IN	denotes	of
T8487	13845-13846	CD	denotes	6
T8489	13847-13849	CC	denotes	or
T8490	13850-13852	CD	denotes	12
T8488	13853-13855	NN	denotes	Gy
T8491	13856-13859	CC	denotes	and
T8492	13860-13869	VBN	denotes	incubated
T8493	13870-13873	IN	denotes	for
T8494	13874-13876	CD	denotes	24
T8495	13877-13878	NN	denotes	h
T8496	13878-13879	.	denotes	.
T8497	13879-14072	sentence	denotes	Cells were then pulse-labeled for 1 h with BrdU (10 μM), fixed in 70% ethanol, double-stained with FITC anti-BrdU and propidium iodide, then sorted by using a Becton Dickinson FACScan machine.
T8498	13880-13885	NNS	denotes	Cells
T8500	13886-13890	VBD	denotes	were
T8501	13891-13895	RB	denotes	then
T8502	13896-13901	NN	denotes	pulse
T8503	13901-13902	HYPH	denotes	-
T8499	13902-13909	VBN	denotes	labeled
T8504	13910-13913	IN	denotes	for
T8505	13914-13915	CD	denotes	1
T8506	13916-13917	NN	denotes	h
T8507	13918-13922	IN	denotes	with
T8508	13923-13927	NN	denotes	BrdU
T8509	13928-13929	-LRB-	denotes	(
T8511	13929-13931	CD	denotes	10
T8510	13932-13934	NN	denotes	μM
T8512	13934-13935	-RRB-	denotes	)
T8513	13935-13937	,	denotes	,
T8514	13937-13942	VBN	denotes	fixed
T8515	13943-13945	IN	denotes	in
T8516	13946-13948	CD	denotes	70
T8517	13948-13949	NN	denotes	%
T8518	13950-13957	NN	denotes	ethanol
T8519	13957-13959	,	denotes	,
T8520	13959-13965	JJ	denotes	double
T8522	13965-13966	HYPH	denotes	-
T8521	13966-13973	VBN	denotes	stained
T8523	13974-13978	IN	denotes	with
T8524	13979-13983	NN	denotes	FITC
T8525	13984-13993	NN	denotes	anti-BrdU
T8526	13994-13997	CC	denotes	and
T8527	13998-14007	NN	denotes	propidium
T8528	14008-14014	NN	denotes	iodide
T8529	14014-14016	,	denotes	,
T8530	14016-14020	RB	denotes	then
T8531	14021-14027	VBN	denotes	sorted
T8532	14028-14030	IN	denotes	by
T8533	14031-14036	VBG	denotes	using
T8534	14037-14038	DT	denotes	a
T8536	14039-14045	NNP	denotes	Becton
T8537	14046-14055	NNP	denotes	Dickinson
T8538	14056-14063	NNP	denotes	FACScan
T8535	14064-14071	NN	denotes	machine
T8539	14071-14072	.	denotes	.
T8540	14072-14129	sentence	denotes	Data were analyzed using Becton Dickinson Cellquest Pro.
T8541	14073-14077	NNS	denotes	Data
T8543	14078-14082	VBD	denotes	were
T8542	14083-14091	VBN	denotes	analyzed
T8544	14092-14097	VBG	denotes	using
T8545	14098-14104	NNP	denotes	Becton
T8546	14105-14114	NNP	denotes	Dickinson
T8548	14115-14124	NNP	denotes	Cellquest
T8547	14125-14128	NNP	denotes	Pro
T8549	14128-14129	.	denotes	.
T11673	14154-14157	DT	denotes	The
T11674	14158-14167	NN	denotes	rationale
T11676	14168-14171	IN	denotes	for
T11677	14172-14176	NN	denotes	RMCE
T11679	14176-14177	HYPH	denotes	-
T11678	14177-14181	NN	denotes	ASAP
T11680	14182-14184	VBZ	denotes	is
T11675	14185-14193	VBN	denotes	detailed
T11681	14194-14196	IN	denotes	in
T11682	14197-14203	NN	denotes	Figure
T11683	14204-14205	CD	denotes	1
T11684	14205-14206	.	denotes	.
T11685	14206-14359	sentence	denotes	The first step requires generating a floxed allele in ES cells that will serve as the substrate for subsequent exchanges (RMCE-ready ES cell, Figure 1).
T11686	14207-14210	DT	denotes	The
T11688	14211-14216	JJ	denotes	first
T11687	14217-14221	NN	denotes	step
T11689	14222-14230	VBZ	denotes	requires
T11690	14231-14241	VBG	denotes	generating
T11691	14242-14243	DT	denotes	a
T11693	14244-14250	VBN	denotes	floxed
T11692	14251-14257	NN	denotes	allele
T11694	14258-14260	IN	denotes	in
T11695	14261-14263	NN	denotes	ES
T11696	14264-14269	NNS	denotes	cells
T11697	14270-14274	WDT	denotes	that
T11699	14275-14279	MD	denotes	will
T11698	14280-14285	VB	denotes	serve
T11700	14286-14288	IN	denotes	as
T11701	14289-14292	DT	denotes	the
T11702	14293-14302	NN	denotes	substrate
T11703	14303-14306	IN	denotes	for
T11704	14307-14317	JJ	denotes	subsequent
T11705	14318-14327	NNS	denotes	exchanges
T11706	14328-14329	-LRB-	denotes	(
T11708	14329-14333	NN	denotes	RMCE
T11710	14333-14334	HYPH	denotes	-
T11709	14334-14339	JJ	denotes	ready
T11712	14340-14342	NN	denotes	ES
T11711	14343-14347	NN	denotes	cell
T11713	14347-14349	,	denotes	,
T11707	14349-14355	NN	denotes	Figure
T11714	14356-14357	CD	denotes	1
T11715	14357-14358	-RRB-	denotes	)
T11716	14358-14359	.	denotes	.
T11717	14359-14407	sentence	denotes	The targeting strategy is detailed in Figure 2.
T11718	14360-14363	DT	denotes	The
T11720	14364-14373	VBG	denotes	targeting
T11719	14374-14382	NN	denotes	strategy
T11722	14383-14385	VBZ	denotes	is
T11721	14386-14394	VBN	denotes	detailed
T11723	14395-14397	IN	denotes	in
T11724	14398-14404	NN	denotes	Figure
T11725	14405-14406	CD	denotes	2
T11726	14406-14407	.	denotes	.
T11727	14407-14534	sentence	denotes	The frequency of targeting was 4% (12/300 puromycin-resistant clones, analyzed by Southern blot and long-range PCR, Figure 2).
T11728	14408-14411	DT	denotes	The
T11729	14412-14421	NN	denotes	frequency
T11731	14422-14424	IN	denotes	of
T11732	14425-14434	NN	denotes	targeting
T11730	14435-14438	VBD	denotes	was
T11733	14439-14440	CD	denotes	4
T11734	14440-14441	NN	denotes	%
T11735	14442-14443	-LRB-	denotes	(
T11737	14443-14445	CD	denotes	12
T11739	14445-14446	HYPH	denotes	/
T11738	14446-14449	CD	denotes	300
T11741	14450-14459	NN	denotes	puromycin
T11743	14459-14460	HYPH	denotes	-
T11742	14460-14469	JJ	denotes	resistant
T11740	14470-14476	NNS	denotes	clones
T11744	14476-14478	,	denotes	,
T11745	14478-14486	VBN	denotes	analyzed
T11746	14487-14489	IN	denotes	by
T11747	14490-14498	NNP	denotes	Southern
T11748	14499-14503	NN	denotes	blot
T11749	14504-14507	CC	denotes	and
T11750	14508-14512	JJ	denotes	long
T11752	14512-14513	HYPH	denotes	-
T11751	14513-14518	NN	denotes	range
T11753	14519-14522	NN	denotes	PCR
T11754	14522-14524	,	denotes	,
T11736	14524-14530	NN	denotes	Figure
T11755	14531-14532	CD	denotes	2
T11756	14532-14533	-RRB-	denotes	)
T11757	14533-14534	.	denotes	.
T11758	14534-14699	sentence	denotes	We next tested the efficiency of RMCE in ES cells, using a replacement construct encoding p53 fused to GFP (p53GFP) to enable tracking p53 in individual live cells.
T11759	14535-14537	PRP	denotes	We
T11761	14538-14542	RB	denotes	next
T11760	14543-14549	VBD	denotes	tested
T11762	14550-14553	DT	denotes	the
T11763	14554-14564	NN	denotes	efficiency
T11764	14565-14567	IN	denotes	of
T11765	14568-14572	NN	denotes	RMCE
T11766	14573-14575	IN	denotes	in
T11767	14576-14578	NN	denotes	ES
T11768	14579-14584	NNS	denotes	cells
T11769	14584-14586	,	denotes	,
T11770	14586-14591	VBG	denotes	using
T11771	14592-14593	DT	denotes	a
T11773	14594-14605	NN	denotes	replacement
T11772	14606-14615	NN	denotes	construct
T11774	14616-14624	VBG	denotes	encoding
T11775	14625-14628	NN	denotes	p53
T11776	14629-14634	VBN	denotes	fused
T11777	14635-14637	IN	denotes	to
T11778	14638-14641	NN	denotes	GFP
T11779	14642-14643	-LRB-	denotes	(
T11780	14643-14649	NN	denotes	p53GFP
T11781	14649-14650	-RRB-	denotes	)
T11782	14651-14653	TO	denotes	to
T11783	14654-14660	VB	denotes	enable
T11784	14661-14669	VBG	denotes	tracking
T11785	14670-14673	NN	denotes	p53
T11786	14674-14676	IN	denotes	in
T11787	14677-14687	JJ	denotes	individual
T11789	14688-14692	JJ	denotes	live
T11788	14693-14698	NNS	denotes	cells
T11790	14698-14699	.	denotes	.
T11791	14699-14866	sentence	denotes	Importantly however, GFP fluorescence was not used to screen cells with targeted events, as we wanted to develop a general method to isolate marker-free recombinants.
T11792	14700-14711	RB	denotes	Importantly
T11794	14712-14719	RB	denotes	however
T11795	14719-14721	,	denotes	,
T11796	14721-14724	NN	denotes	GFP
T11797	14725-14737	NN	denotes	fluorescence
T11798	14738-14741	VBD	denotes	was
T11799	14742-14745	RB	denotes	not
T11793	14746-14750	VBN	denotes	used
T11800	14751-14753	TO	denotes	to
T11801	14754-14760	VB	denotes	screen
T11802	14761-14766	NNS	denotes	cells
T11803	14767-14771	IN	denotes	with
T11804	14772-14780	VBN	denotes	targeted
T11805	14781-14787	NNS	denotes	events
T11806	14787-14789	,	denotes	,
T11807	14789-14791	IN	denotes	as
T11809	14792-14794	PRP	denotes	we
T11808	14795-14801	VBD	denotes	wanted
T11810	14802-14804	TO	denotes	to
T11811	14805-14812	VB	denotes	develop
T11812	14813-14814	DT	denotes	a
T11814	14815-14822	JJ	denotes	general
T11813	14823-14829	NN	denotes	method
T11815	14830-14832	TO	denotes	to
T11816	14833-14840	VB	denotes	isolate
T11817	14841-14847	NN	denotes	marker
T11819	14847-14848	HYPH	denotes	-
T11818	14848-14852	JJ	denotes	free
T11820	14853-14865	NNS	denotes	recombinants
T11821	14865-14866	.	denotes	.
T11822	14866-14914	sentence	denotes	The exchange strategy is detailed in Figure 3A.
T11823	14867-14870	DT	denotes	The
T11825	14871-14879	NN	denotes	exchange
T11824	14880-14888	NN	denotes	strategy
T11827	14889-14891	VBZ	denotes	is
T11826	14892-14900	VBN	denotes	detailed
T11828	14901-14903	IN	denotes	in
T11829	14904-14910	NN	denotes	Figure
T11830	14911-14913	NN	denotes	3A
T11831	14913-14914	.	denotes	.
T11832	14914-15085	sentence	denotes	We picked 65 ES cell clones resistant to 1-(-2-deoxy-2-fluoro-1-b-d-arabino-furanosyl)-5-iodouracil (FIAU) due to TK loss and analyzed their DNA by PCR and Southern blot.
T11833	14915-14917	PRP	denotes	We
T11834	14918-14924	VBD	denotes	picked
T11835	14925-14927	CD	denotes	65
T11837	14928-14930	NN	denotes	ES
T11838	14931-14935	NN	denotes	cell
T11836	14936-14942	NNS	denotes	clones
T11839	14943-14952	JJ	denotes	resistant
T11840	14953-14955	IN	denotes	to
T11841	14956-14957	CD	denotes	1
T11843	14957-14958	HYPH	denotes	-
T11844	14958-14959	-LRB-	denotes	(
T11846	14959-14960	HYPH	denotes	-
T11847	14960-14961	CD	denotes	2
T11848	14961-14962	HYPH	denotes	-
T11849	14962-14967	NN	denotes	deoxy
T11850	14967-14968	HYPH	denotes	-
T11851	14968-14969	CD	denotes	2
T11852	14969-14970	HYPH	denotes	-
T11853	14970-14976	NN	denotes	fluoro
T11854	14976-14977	HYPH	denotes	-
T11855	14977-14978	CD	denotes	1
T11856	14978-14979	HYPH	denotes	-
T11857	14979-14980	NN	denotes	b
T11858	14980-14981	HYPH	denotes	-
T11859	14981-14982	NN	denotes	d
T11860	14982-14983	HYPH	denotes	-
T11861	14983-14990	NN	denotes	arabino
T11862	14990-14991	HYPH	denotes	-
T11845	14991-15000	NN	denotes	furanosyl
T11863	15000-15001	-RRB-	denotes	)
T11864	15001-15002	HYPH	denotes	-
T11842	15002-15003	CD	denotes	5
T11866	15003-15004	HYPH	denotes	-
T11865	15004-15014	NN	denotes	iodouracil
T11867	15015-15016	-LRB-	denotes	(
T11868	15016-15020	NN	denotes	FIAU
T11869	15020-15021	-RRB-	denotes	)
T11870	15022-15025	IN	denotes	due
T11871	15026-15028	IN	denotes	to
T11872	15029-15031	NN	denotes	TK
T11873	15032-15036	NN	denotes	loss
T11874	15037-15040	CC	denotes	and
T11875	15041-15049	VBD	denotes	analyzed
T11876	15050-15055	PRP$	denotes	their
T11877	15056-15059	NN	denotes	DNA
T11878	15060-15062	IN	denotes	by
T11879	15063-15066	NN	denotes	PCR
T11880	15067-15070	CC	denotes	and
T11881	15071-15079	NNP	denotes	Southern
T11882	15080-15084	NN	denotes	blot
T11883	15084-15085	.	denotes	.
T11884	15085-15181	sentence	denotes	Strikingly, 54 proper recombinants were identified, indicating very high RMCE efficiency (83%).
T11885	15086-15096	RB	denotes	Strikingly
T11887	15096-15098	,	denotes	,
T11888	15098-15100	CD	denotes	54
T11890	15101-15107	JJ	denotes	proper
T11889	15108-15120	NNS	denotes	recombinants
T11891	15121-15125	VBD	denotes	were
T11886	15126-15136	VBN	denotes	identified
T11892	15136-15138	,	denotes	,
T11893	15138-15148	VBG	denotes	indicating
T11894	15149-15153	RB	denotes	very
T11895	15154-15158	JJ	denotes	high
T11897	15159-15163	NN	denotes	RMCE
T11896	15164-15174	NN	denotes	efficiency
T11898	15175-15176	-LRB-	denotes	(
T11900	15176-15178	CD	denotes	83
T11899	15178-15179	NN	denotes	%
T11901	15179-15180	-RRB-	denotes	)
T11902	15180-15181	.	denotes	.
T11903	15181-15287	sentence	denotes	RMCE also proved to be precise, as no aberrant bands were detected in PCR and Southern blots (Figure 3A).
T11904	15182-15186	NN	denotes	RMCE
T11906	15187-15191	RB	denotes	also
T11905	15192-15198	VBD	denotes	proved
T11907	15199-15201	TO	denotes	to
T11908	15202-15204	VB	denotes	be
T11909	15205-15212	JJ	denotes	precise
T11910	15212-15214	,	denotes	,
T11911	15214-15216	IN	denotes	as
T11913	15217-15219	DT	denotes	no
T11915	15220-15228	JJ	denotes	aberrant
T11914	15229-15234	NNS	denotes	bands
T11916	15235-15239	VBD	denotes	were
T11912	15240-15248	VBN	denotes	detected
T11917	15249-15251	IN	denotes	in
T11918	15252-15255	NN	denotes	PCR
T11919	15256-15259	CC	denotes	and
T11920	15260-15268	NNP	denotes	Southern
T11921	15269-15274	NNS	denotes	blots
T11922	15275-15276	-LRB-	denotes	(
T11924	15276-15282	NN	denotes	Figure
T11923	15283-15285	NN	denotes	3A
T11925	15285-15286	-RRB-	denotes	)
T11926	15286-15287	.	denotes	.
T11927	15287-15441	sentence	denotes	p53+/GFP ES clones were analyzed by western blot with an antibody against p53, and found to express an additional band at the expected size (ca. 80 kDa).
T11928	15288-15291	NN	denotes	p53
T11930	15291-15292	SYM	denotes	+
T11931	15292-15293	HYPH	denotes	/
T11929	15293-15296	NN	denotes	GFP
T11933	15297-15299	NN	denotes	ES
T11932	15300-15306	NNS	denotes	clones
T11935	15307-15311	VBD	denotes	were
T11934	15312-15320	VBN	denotes	analyzed
T11936	15321-15323	IN	denotes	by
T11937	15324-15331	NNP	denotes	western
T11938	15332-15336	NN	denotes	blot
T11939	15337-15341	IN	denotes	with
T11940	15342-15344	DT	denotes	an
T11941	15345-15353	NN	denotes	antibody
T11942	15354-15361	IN	denotes	against
T11943	15362-15365	NN	denotes	p53
T11944	15365-15367	,	denotes	,
T11945	15367-15370	CC	denotes	and
T11946	15371-15376	VBN	denotes	found
T11947	15377-15379	TO	denotes	to
T11948	15380-15387	VB	denotes	express
T11949	15388-15390	DT	denotes	an
T11951	15391-15401	JJ	denotes	additional
T11950	15402-15406	NN	denotes	band
T11952	15407-15409	IN	denotes	at
T11953	15410-15413	DT	denotes	the
T11955	15414-15422	VBN	denotes	expected
T11954	15423-15427	NN	denotes	size
T11956	15428-15429	-LRB-	denotes	(
T11957	15429-15432	NN	denotes	ca.
T11958	15433-15435	CD	denotes	80
T11959	15436-15439	NN	denotes	kDa
T11960	15439-15440	-RRB-	denotes	)
T11961	15440-15441	.	denotes	.
T11962	15441-15765	sentence	denotes	Surprisingly, the fusion of GFP to p53 apparently altered p53 stability: steady-state levels of p53GFP were much higher than those of wild-type p53 (p53WT) in unstressed cells, and did not vary significantly after DNA damage, so that the levels for both p53WT and p53GFP were similar after adriamycin treatment (Figure 3A).
T11963	15442-15454	RB	denotes	Surprisingly
T11965	15454-15456	,	denotes	,
T11966	15456-15459	DT	denotes	the
T11967	15460-15466	NN	denotes	fusion
T11968	15467-15469	IN	denotes	of
T11969	15470-15473	NN	denotes	GFP
T11970	15474-15476	IN	denotes	to
T11971	15477-15480	NN	denotes	p53
T11972	15481-15491	RB	denotes	apparently
T11964	15492-15499	VBD	denotes	altered
T11974	15500-15503	NN	denotes	p53
T11975	15504-15513	NN	denotes	stability
T11976	15513-15515	:	denotes	:
T11977	15515-15521	JJ	denotes	steady
T11979	15521-15522	HYPH	denotes	-
T11978	15522-15527	NN	denotes	state
T11980	15528-15534	NNS	denotes	levels
T11981	15535-15537	IN	denotes	of
T11982	15538-15544	NN	denotes	p53GFP
T11973	15545-15549	VBD	denotes	were
T11983	15550-15554	RB	denotes	much
T11984	15555-15561	JJR	denotes	higher
T11985	15562-15566	IN	denotes	than
T11986	15567-15572	DT	denotes	those
T11987	15573-15575	IN	denotes	of
T11988	15576-15580	JJ	denotes	wild
T11990	15580-15581	HYPH	denotes	-
T11989	15581-15585	NN	denotes	type
T11991	15586-15589	NN	denotes	p53
T11992	15590-15591	-LRB-	denotes	(
T11993	15591-15596	NN	denotes	p53WT
T11994	15596-15597	-RRB-	denotes	)
T11995	15598-15600	IN	denotes	in
T11996	15601-15611	JJ	denotes	unstressed
T11997	15612-15617	NNS	denotes	cells
T11998	15617-15619	,	denotes	,
T11999	15619-15622	CC	denotes	and
T12000	15623-15626	VBD	denotes	did
T12002	15627-15630	RB	denotes	not
T12001	15631-15635	VB	denotes	vary
T12003	15636-15649	RB	denotes	significantly
T12004	15650-15655	IN	denotes	after
T12005	15656-15659	NN	denotes	DNA
T12006	15660-15666	NN	denotes	damage
T12007	15666-15668	,	denotes	,
T12008	15668-15670	IN	denotes	so
T12010	15671-15675	IN	denotes	that
T12011	15676-15679	DT	denotes	the
T12012	15680-15686	NNS	denotes	levels
T12013	15687-15690	IN	denotes	for
T12014	15691-15695	CC	denotes	both
T12015	15696-15701	NN	denotes	p53WT
T12016	15702-15705	CC	denotes	and
T12017	15706-15712	NN	denotes	p53GFP
T12009	15713-15717	VBD	denotes	were
T12018	15718-15725	JJ	denotes	similar
T12019	15726-15731	IN	denotes	after
T12020	15732-15742	NN	denotes	adriamycin
T12021	15743-15752	NN	denotes	treatment
T12022	15753-15754	-LRB-	denotes	(
T12024	15754-15760	NN	denotes	Figure
T12023	15761-15763	NN	denotes	3A
T12025	15763-15764	-RRB-	denotes	)
T12026	15764-15765	.	denotes	.
T12027	15765-15898	sentence	denotes	Six independent p53+/GFP clones were injected into blastocysts and transferred to pseudo-pregnant females using standard procedures.
T12028	15766-15769	CD	denotes	Six
T12030	15770-15781	JJ	denotes	independent
T12031	15782-15785	NN	denotes	p53
T12033	15785-15786	SYM	denotes	+
T12034	15786-15787	HYPH	denotes	/
T12032	15787-15790	NN	denotes	GFP
T12029	15791-15797	NNS	denotes	clones
T12036	15798-15802	VBD	denotes	were
T12035	15803-15811	VBN	denotes	injected
T12037	15812-15816	IN	denotes	into
T12038	15817-15828	NNS	denotes	blastocysts
T12039	15829-15832	CC	denotes	and
T12040	15833-15844	VBN	denotes	transferred
T12041	15845-15847	IN	denotes	to
T12042	15848-15854	JJ	denotes	pseudo
T12044	15854-15855	HYPH	denotes	-
T12043	15855-15863	JJ	denotes	pregnant
T12045	15864-15871	NNS	denotes	females
T12046	15872-15877	VBG	denotes	using
T12047	15878-15886	JJ	denotes	standard
T12048	15887-15897	NNS	denotes	procedures
T12049	15897-15898	.	denotes	.
T12050	15898-15940	sentence	denotes	Strikingly, no pregnancies were obtained.
T12051	15899-15909	RB	denotes	Strikingly
T12053	15909-15911	,	denotes	,
T12054	15911-15913	DT	denotes	no
T12055	15914-15925	NNS	denotes	pregnancies
T12056	15926-15930	VBD	denotes	were
T12052	15931-15939	VBN	denotes	obtained
T12057	15939-15940	.	denotes	.
T12058	15940-16154	sentence	denotes	It has been shown that the p53 pathway is regulated very differently in ES and somatic cells: ES cells contain relatively high p53 levels and lack the p53-mediated DNA damage responses found in somatic cells (18).
T12059	15941-15943	PRP	denotes	It
T12061	15944-15947	VBZ	denotes	has
T12062	15948-15952	VBN	denotes	been
T12060	15953-15958	VBN	denotes	shown
T12064	15959-15963	IN	denotes	that
T12066	15964-15967	DT	denotes	the
T12068	15968-15971	NN	denotes	p53
T12067	15972-15979	NN	denotes	pathway
T12069	15980-15982	VBZ	denotes	is
T12065	15983-15992	VBN	denotes	regulated
T12070	15993-15997	RB	denotes	very
T12071	15998-16009	RB	denotes	differently
T12072	16010-16012	IN	denotes	in
T12073	16013-16015	NN	denotes	ES
T12075	16016-16019	CC	denotes	and
T12076	16020-16027	JJ	denotes	somatic
T12074	16028-16033	NNS	denotes	cells
T12077	16033-16035	:	denotes	:
T12078	16035-16037	NN	denotes	ES
T12079	16038-16043	NNS	denotes	cells
T12063	16044-16051	VBP	denotes	contain
T12080	16052-16062	RB	denotes	relatively
T12081	16063-16067	JJ	denotes	high
T12083	16068-16071	NN	denotes	p53
T12082	16072-16078	NNS	denotes	levels
T12084	16079-16082	CC	denotes	and
T12085	16083-16087	VBP	denotes	lack
T12086	16088-16091	DT	denotes	the
T12088	16092-16095	NN	denotes	p53
T12089	16095-16096	HYPH	denotes	-
T12090	16096-16104	JJ	denotes	mediated
T12091	16105-16108	NN	denotes	DNA
T12092	16109-16115	NN	denotes	damage
T12087	16116-16125	NNS	denotes	responses
T12093	16126-16131	VBN	denotes	found
T12094	16132-16134	IN	denotes	in
T12095	16135-16142	JJ	denotes	somatic
T12096	16143-16148	NNS	denotes	cells
T12097	16149-16150	-LRB-	denotes	(
T12098	16150-16152	CD	denotes	18
T12099	16152-16153	-RRB-	denotes	)
T12100	16153-16154	.	denotes	.
T12101	16154-16526	sentence	denotes	This, together with the observation that p53 levels decrease during mouse embryogenesis (19), suggested an explanation for the observed lack of pregnancies: we speculate that the high levels of p53GFP in the ES cells injected into blastocysts might have prevented normal embryonic development once these cells began to differentiate and the p53 pathway became functional.
T12102	16155-16159	DT	denotes	This
T12104	16159-16161	,	denotes	,
T12105	16161-16169	RB	denotes	together
T12106	16170-16174	IN	denotes	with
T12107	16175-16178	DT	denotes	the
T12108	16179-16190	NN	denotes	observation
T12109	16191-16195	IN	denotes	that
T12111	16196-16199	NN	denotes	p53
T12112	16200-16206	NNS	denotes	levels
T12110	16207-16215	VBP	denotes	decrease
T12113	16216-16222	IN	denotes	during
T12114	16223-16228	NN	denotes	mouse
T12115	16229-16242	NN	denotes	embryogenesis
T12116	16243-16244	-LRB-	denotes	(
T12117	16244-16246	CD	denotes	19
T12118	16246-16247	-RRB-	denotes	)
T12119	16247-16249	,	denotes	,
T12103	16249-16258	VBD	denotes	suggested
T12121	16259-16261	DT	denotes	an
T12122	16262-16273	NN	denotes	explanation
T12123	16274-16277	IN	denotes	for
T12124	16278-16281	DT	denotes	the
T12126	16282-16290	VBN	denotes	observed
T12125	16291-16295	NN	denotes	lack
T12127	16296-16298	IN	denotes	of
T12128	16299-16310	NNS	denotes	pregnancies
T12129	16310-16312	:	denotes	:
T12130	16312-16314	PRP	denotes	we
T12120	16315-16324	VBP	denotes	speculate
T12131	16325-16329	IN	denotes	that
T12133	16330-16333	DT	denotes	the
T12135	16334-16338	JJ	denotes	high
T12134	16339-16345	NNS	denotes	levels
T12136	16346-16348	IN	denotes	of
T12137	16349-16355	NN	denotes	p53GFP
T12138	16356-16358	IN	denotes	in
T12139	16359-16362	DT	denotes	the
T12141	16363-16365	NN	denotes	ES
T12140	16366-16371	NNS	denotes	cells
T12142	16372-16380	VBN	denotes	injected
T12143	16381-16385	IN	denotes	into
T12144	16386-16397	NNS	denotes	blastocysts
T12145	16398-16403	MD	denotes	might
T12146	16404-16408	VB	denotes	have
T12132	16409-16418	VBN	denotes	prevented
T12147	16419-16425	JJ	denotes	normal
T12149	16426-16435	JJ	denotes	embryonic
T12148	16436-16447	NN	denotes	development
T12150	16448-16452	IN	denotes	once
T12152	16453-16458	DT	denotes	these
T12153	16459-16464	NNS	denotes	cells
T12151	16465-16470	VBD	denotes	began
T12154	16471-16473	TO	denotes	to
T12155	16474-16487	VB	denotes	differentiate
T12156	16488-16491	CC	denotes	and
T12157	16492-16495	DT	denotes	the
T12159	16496-16499	NN	denotes	p53
T12158	16500-16507	NN	denotes	pathway
T12160	16508-16514	VBD	denotes	became
T12161	16515-16525	JJ	denotes	functional
T12162	16525-16526	.	denotes	.
T12163	16526-16689	sentence	denotes	To test this possibility, we performed RMCE with a p53 fusion gene in which the p53 proline-rich domain (PRD) was deleted (p53ΔP), and the p53ΔP was fused to GFP.
T12164	16527-16529	TO	denotes	To
T12165	16530-16534	VB	denotes	test
T12167	16535-16539	DT	denotes	this
T12168	16540-16551	NN	denotes	possibility
T12169	16551-16553	,	denotes	,
T12170	16553-16555	PRP	denotes	we
T12166	16556-16565	VBD	denotes	performed
T12171	16566-16570	NN	denotes	RMCE
T12172	16571-16575	IN	denotes	with
T12173	16576-16577	DT	denotes	a
T12175	16578-16581	NN	denotes	p53
T12176	16582-16588	NN	denotes	fusion
T12174	16589-16593	NN	denotes	gene
T12177	16594-16596	IN	denotes	in
T12179	16597-16602	WDT	denotes	which
T12180	16603-16606	DT	denotes	the
T12182	16607-16610	NN	denotes	p53
T12183	16611-16618	NN	denotes	proline
T12185	16618-16619	HYPH	denotes	-
T12184	16619-16623	JJ	denotes	rich
T12181	16624-16630	NN	denotes	domain
T12186	16631-16632	-LRB-	denotes	(
T12187	16632-16635	NN	denotes	PRD
T12188	16635-16636	-RRB-	denotes	)
T12189	16637-16640	VBD	denotes	was
T12178	16641-16648	VBN	denotes	deleted
T12190	16649-16650	-LRB-	denotes	(
T12191	16650-16655	NN	denotes	p53ΔP
T12192	16655-16656	-RRB-	denotes	)
T12193	16656-16658	,	denotes	,
T12194	16658-16661	CC	denotes	and
T12195	16662-16665	DT	denotes	the
T12196	16666-16671	NN	denotes	p53ΔP
T12198	16672-16675	VBD	denotes	was
T12197	16676-16681	VBN	denotes	fused
T12199	16682-16684	IN	denotes	to
T12200	16685-16688	NN	denotes	GFP
T12201	16688-16689	.	denotes	.
T12202	16689-16820	sentence	denotes	We used this mutant because deleting the proline-rich domain decreases stability and compromises DNA-damage responses in vivo (5).
T12203	16690-16692	PRP	denotes	We
T12204	16693-16697	VBD	denotes	used
T12205	16698-16702	DT	denotes	this
T12206	16703-16709	NN	denotes	mutant
T12207	16710-16717	IN	denotes	because
T12209	16718-16726	VBG	denotes	deleting
T12210	16727-16730	DT	denotes	the
T12212	16731-16738	NN	denotes	proline
T12214	16738-16739	HYPH	denotes	-
T12213	16739-16743	JJ	denotes	rich
T12211	16744-16750	NN	denotes	domain
T12208	16751-16760	VBZ	denotes	decreases
T12215	16761-16770	NN	denotes	stability
T12216	16771-16774	CC	denotes	and
T12217	16775-16786	VBZ	denotes	compromises
T12218	16787-16790	NN	denotes	DNA
T12220	16790-16791	HYPH	denotes	-
T12219	16791-16797	NN	denotes	damage
T12221	16798-16807	NNS	denotes	responses
T12222	16808-16810	FW	denotes	in
T12223	16811-16815	FW	denotes	vivo
T12224	16816-16817	-LRB-	denotes	(
T12225	16817-16818	CD	denotes	5
T12226	16818-16819	-RRB-	denotes	)
T12227	16819-16820	.	denotes	.
T12228	16820-16915	sentence	denotes	According to our hypothesis, this hypomorphic mutant should not prevent embryonic development.
T12229	16821-16830	VBG	denotes	According
T12231	16831-16833	IN	denotes	to
T12232	16834-16837	PRP$	denotes	our
T12233	16838-16848	NN	denotes	hypothesis
T12234	16848-16850	,	denotes	,
T12235	16850-16854	DT	denotes	this
T12237	16855-16866	JJ	denotes	hypomorphic
T12236	16867-16873	NN	denotes	mutant
T12238	16874-16880	MD	denotes	should
T12239	16881-16884	RB	denotes	not
T12230	16885-16892	VB	denotes	prevent
T12240	16893-16902	JJ	denotes	embryonic
T12241	16903-16914	NN	denotes	development
T12242	16914-16915	.	denotes	.
T12243	16915-17102	sentence	denotes	RMCE with a p53ΔPGFP replacement plasmid was again very efficient and western blots revealed an additional band of the predicted molecular weight only in p53+/ΔPGFP ES cells (Figure 3B).
T12244	16916-16920	NN	denotes	RMCE
T12246	16921-16925	IN	denotes	with
T12247	16926-16927	DT	denotes	a
T12249	16928-16936	NN	denotes	p53ΔPGFP
T12250	16937-16948	NN	denotes	replacement
T12248	16949-16956	NN	denotes	plasmid
T12245	16957-16960	VBD	denotes	was
T12251	16961-16966	RB	denotes	again
T12252	16967-16971	RB	denotes	very
T12253	16972-16981	JJ	denotes	efficient
T12254	16982-16985	CC	denotes	and
T12255	16986-16993	NNP	denotes	western
T12256	16994-16999	NNS	denotes	blots
T12257	17000-17008	VBD	denotes	revealed
T12258	17009-17011	DT	denotes	an
T12260	17012-17022	JJ	denotes	additional
T12259	17023-17027	NN	denotes	band
T12261	17028-17030	IN	denotes	of
T12262	17031-17034	DT	denotes	the
T12264	17035-17044	VBN	denotes	predicted
T12265	17045-17054	JJ	denotes	molecular
T12263	17055-17061	NN	denotes	weight
T12266	17062-17066	RB	denotes	only
T12267	17067-17069	IN	denotes	in
T12268	17070-17073	NN	denotes	p53
T12270	17073-17074	SYM	denotes	+
T12271	17074-17075	HYPH	denotes	/
T12269	17075-17080	NN	denotes	ΔPGFP
T12273	17081-17083	NN	denotes	ES
T12272	17084-17089	NNS	denotes	cells
T12274	17090-17091	-LRB-	denotes	(
T12276	17091-17097	NN	denotes	Figure
T12275	17098-17100	NN	denotes	3B
T12277	17100-17101	-RRB-	denotes	)
T12278	17101-17102	.	denotes	.
T12279	17102-17254	sentence	denotes	As expected, the PRD deletion correlated with lower expression levels: p53ΔPGFP was much less abundant than p53WT in all p53+/ΔPGFP clones (Figure 3B).
T12280	17103-17105	IN	denotes	As
T12281	17106-17114	VBN	denotes	expected
T12283	17114-17116	,	denotes	,
T12284	17116-17119	DT	denotes	the
T12286	17120-17123	NN	denotes	PRD
T12285	17124-17132	NN	denotes	deletion
T12282	17133-17143	VBD	denotes	correlated
T12288	17144-17148	IN	denotes	with
T12289	17149-17154	JJR	denotes	lower
T12291	17155-17165	NN	denotes	expression
T12290	17166-17172	NNS	denotes	levels
T12292	17172-17174	:	denotes	:
T12293	17174-17182	NN	denotes	p53ΔPGFP
T12287	17183-17186	VBD	denotes	was
T12294	17187-17191	RB	denotes	much
T12295	17192-17196	RBR	denotes	less
T12296	17197-17205	JJ	denotes	abundant
T12297	17206-17210	IN	denotes	than
T12298	17211-17216	NN	denotes	p53WT
T12299	17217-17219	IN	denotes	in
T12300	17220-17223	DT	denotes	all
T12302	17224-17227	NN	denotes	p53
T12304	17227-17228	SYM	denotes	+
T12305	17228-17229	HYPH	denotes	/
T12303	17229-17234	NN	denotes	ΔPGFP
T12301	17235-17241	NNS	denotes	clones
T12306	17242-17243	-LRB-	denotes	(
T12308	17243-17249	NN	denotes	Figure
T12307	17250-17252	NN	denotes	3B
T12309	17252-17253	-RRB-	denotes	)
T12310	17253-17254	.	denotes	.
T12311	17254-17385	sentence	denotes	We next determined whether p53+/ΔPGFP ES cells could generate chimeric mice and transmit the modified allele through the germline.
T12312	17255-17257	PRP	denotes	We
T12314	17258-17262	RB	denotes	next
T12313	17263-17273	VBD	denotes	determined
T12315	17274-17281	IN	denotes	whether
T12317	17282-17285	NN	denotes	p53
T12319	17285-17286	SYM	denotes	+
T12320	17286-17287	HYPH	denotes	/
T12318	17287-17292	NN	denotes	ΔPGFP
T12322	17293-17295	NN	denotes	ES
T12321	17296-17301	NNS	denotes	cells
T12323	17302-17307	MD	denotes	could
T12316	17308-17316	VB	denotes	generate
T12324	17317-17325	JJ	denotes	chimeric
T12325	17326-17330	NNS	denotes	mice
T12326	17331-17334	CC	denotes	and
T12327	17335-17343	VB	denotes	transmit
T12328	17344-17347	DT	denotes	the
T12330	17348-17356	VBN	denotes	modified
T12329	17357-17363	NN	denotes	allele
T12331	17364-17371	IN	denotes	through
T12332	17372-17375	DT	denotes	the
T12333	17376-17384	NN	denotes	germline
T12334	17384-17385	.	denotes	.
T12335	17385-17493	sentence	denotes	Two p53+/ΔPGFP ES cell clones were injected into blastocysts and highly chimeric (>80%) mice were obtained.
T12336	17386-17389	CD	denotes	Two
T12338	17390-17393	NN	denotes	p53
T12340	17393-17394	SYM	denotes	+
T12341	17394-17395	HYPH	denotes	/
T12339	17395-17400	NN	denotes	ΔPGFP
T12342	17401-17403	NN	denotes	ES
T12343	17404-17408	NN	denotes	cell
T12337	17409-17415	NNS	denotes	clones
T12345	17416-17420	VBD	denotes	were
T12344	17421-17429	VBN	denotes	injected
T12346	17430-17434	IN	denotes	into
T12347	17435-17446	NNS	denotes	blastocysts
T12348	17447-17450	CC	denotes	and
T12349	17451-17457	RB	denotes	highly
T12350	17458-17466	JJ	denotes	chimeric
T12352	17467-17468	-LRB-	denotes	(
T12353	17468-17469	SYM	denotes	>
T12354	17469-17471	CD	denotes	80
T12351	17471-17472	NN	denotes	%
T12356	17472-17473	-RRB-	denotes	)
T12355	17474-17478	NNS	denotes	mice
T12358	17479-17483	VBD	denotes	were
T12357	17484-17492	VBN	denotes	obtained
T12359	17492-17493	.	denotes	.
T12360	17493-17581	sentence	denotes	Heterozygote pups were recovered from the mating the chimeras with WT mice (Figure 3C).
T12361	17494-17506	NN	denotes	Heterozygote
T12362	17507-17511	NNS	denotes	pups
T12364	17512-17516	VBD	denotes	were
T12363	17517-17526	VBN	denotes	recovered
T12365	17527-17531	IN	denotes	from
T12366	17532-17535	DT	denotes	the
T12367	17536-17542	NN	denotes	mating
T12368	17543-17546	DT	denotes	the
T12369	17547-17555	NNS	denotes	chimeras
T12370	17556-17560	IN	denotes	with
T12371	17561-17563	NN	denotes	WT
T12372	17564-17568	NNS	denotes	mice
T12373	17569-17570	-LRB-	denotes	(
T12375	17570-17576	NN	denotes	Figure
T12374	17577-17579	NN	denotes	3C
T12376	17579-17580	-RRB-	denotes	)
T12377	17580-17581	.	denotes	.
T12378	17581-17736	sentence	denotes	These data demonstrate that marker-free RMCE is very efficient in ES cells and allows germline transmission of a targeted mutation (see Figure 1, path A).
T12379	17582-17587	DT	denotes	These
T12380	17588-17592	NNS	denotes	data
T12381	17593-17604	VBP	denotes	demonstrate
T12382	17605-17609	IN	denotes	that
T12384	17610-17616	NN	denotes	marker
T12386	17616-17617	HYPH	denotes	-
T12385	17617-17621	JJ	denotes	free
T12387	17622-17626	NN	denotes	RMCE
T12383	17627-17629	VBZ	denotes	is
T12388	17630-17634	RB	denotes	very
T12389	17635-17644	JJ	denotes	efficient
T12390	17645-17647	IN	denotes	in
T12391	17648-17650	NN	denotes	ES
T12392	17651-17656	NNS	denotes	cells
T12393	17657-17660	CC	denotes	and
T12394	17661-17667	VBZ	denotes	allows
T12395	17668-17676	NN	denotes	germline
T12396	17677-17689	NN	denotes	transmission
T12397	17690-17692	IN	denotes	of
T12398	17693-17694	DT	denotes	a
T12400	17695-17703	VBN	denotes	targeted
T12399	17704-17712	NN	denotes	mutation
T12401	17713-17714	-LRB-	denotes	(
T12402	17714-17717	VB	denotes	see
T12403	17718-17724	NN	denotes	Figure
T12404	17725-17726	CD	denotes	1
T12405	17726-17728	,	denotes	,
T12406	17728-17732	NN	denotes	path
T12407	17733-17734	NN	denotes	A
T12408	17734-17735	-RRB-	denotes	)
T12409	17735-17736	.	denotes	.
T12410	17736-17870	sentence	denotes	We next determined whether the RMCE approach could be used to target mutations at the p53 allele in somatic cells (Figure 1, path B).
T12411	17737-17739	PRP	denotes	We
T12413	17740-17744	RB	denotes	next
T12412	17745-17755	VBD	denotes	determined
T12414	17756-17763	IN	denotes	whether
T12416	17764-17767	DT	denotes	the
T12418	17768-17772	NN	denotes	RMCE
T12417	17773-17781	NN	denotes	approach
T12419	17782-17787	MD	denotes	could
T12420	17788-17790	VB	denotes	be
T12415	17791-17795	VBN	denotes	used
T12421	17796-17798	TO	denotes	to
T12422	17799-17805	VB	denotes	target
T12423	17806-17815	NNS	denotes	mutations
T12424	17816-17818	IN	denotes	at
T12425	17819-17822	DT	denotes	the
T12427	17823-17826	NN	denotes	p53
T12426	17827-17833	NN	denotes	allele
T12428	17834-17836	IN	denotes	in
T12429	17837-17844	JJ	denotes	somatic
T12430	17845-17850	NNS	denotes	cells
T12431	17851-17852	-LRB-	denotes	(
T12432	17852-17858	NN	denotes	Figure
T12433	17859-17860	CD	denotes	1
T12434	17860-17862	,	denotes	,
T12435	17862-17866	NN	denotes	path
T12436	17867-17868	NN	denotes	B
T12437	17868-17869	-RRB-	denotes	)
T12438	17869-17870	.	denotes	.
T12439	17870-18033	sentence	denotes	We first verified that the RMCE-ready p53 locus (p53RMCE) could be transmitted through the germline by mating p53RMCE/+ chimeras with p53+/− mice (20) (Figure 4).
T12440	17871-17873	PRP	denotes	We
T12442	17874-17879	RB	denotes	first
T12441	17880-17888	VBD	denotes	verified
T12443	17889-17893	IN	denotes	that
T12445	17894-17897	DT	denotes	the
T12447	17898-17902	NN	denotes	RMCE
T12449	17902-17903	HYPH	denotes	-
T12448	17903-17908	JJ	denotes	ready
T12450	17909-17912	NN	denotes	p53
T12446	17913-17918	NN	denotes	locus
T12451	17919-17920	-LRB-	denotes	(
T12452	17920-17927	NN	denotes	p53RMCE
T12453	17927-17928	-RRB-	denotes	)
T12454	17929-17934	MD	denotes	could
T12455	17935-17937	VB	denotes	be
T12444	17938-17949	VBN	denotes	transmitted
T12456	17950-17957	IN	denotes	through
T12457	17958-17961	DT	denotes	the
T12458	17962-17970	NN	denotes	germline
T12459	17971-17973	IN	denotes	by
T12460	17974-17980	VBG	denotes	mating
T12461	17981-17988	NN	denotes	p53RMCE
T12463	17988-17989	HYPH	denotes	/
T12464	17989-17990	SYM	denotes	+
T12462	17991-17999	NNS	denotes	chimeras
T12465	18000-18004	IN	denotes	with
T12466	18005-18008	NN	denotes	p53
T12468	18008-18009	SYM	denotes	+
T12469	18009-18010	HYPH	denotes	/
T12470	18010-18011	SYM	denotes	−
T12467	18012-18016	NNS	denotes	mice
T12471	18017-18018	-LRB-	denotes	(
T12472	18018-18020	CD	denotes	20
T12473	18020-18021	-RRB-	denotes	)
T12474	18022-18023	-LRB-	denotes	(
T12475	18023-18029	NN	denotes	Figure
T12476	18030-18031	CD	denotes	4
T12477	18031-18032	-RRB-	denotes	)
T12478	18032-18033	.	denotes	.
T12479	18033-18154	sentence	denotes	Importantly, this allowed us to generate p53RMCE/− MEFs, which were used to test RMCE at the p53 locus in somatic cells.
T12480	18034-18045	RB	denotes	Importantly
T12482	18045-18047	,	denotes	,
T12483	18047-18051	DT	denotes	this
T12481	18052-18059	VBD	denotes	allowed
T12484	18060-18062	PRP	denotes	us
T12486	18063-18065	TO	denotes	to
T12485	18066-18074	VB	denotes	generate
T12487	18075-18082	NN	denotes	p53RMCE
T12489	18082-18083	HYPH	denotes	/
T12490	18083-18084	SYM	denotes	−
T12488	18085-18089	NNS	denotes	MEFs
T12491	18089-18091	,	denotes	,
T12492	18091-18096	WDT	denotes	which
T12494	18097-18101	VBD	denotes	were
T12493	18102-18106	VBN	denotes	used
T12495	18107-18109	TO	denotes	to
T12496	18110-18114	VB	denotes	test
T12497	18115-18119	NN	denotes	RMCE
T12498	18120-18122	IN	denotes	at
T12499	18123-18126	DT	denotes	the
T12501	18127-18130	NN	denotes	p53
T12500	18131-18136	NN	denotes	locus
T12502	18137-18139	IN	denotes	in
T12503	18140-18147	JJ	denotes	somatic
T12504	18148-18153	NNS	denotes	cells
T12505	18153-18154	.	denotes	.
T12506	18154-18322	sentence	denotes	We first attempted RMCE in MEFs by electroporating p53RMCE/− MEFs with a Cre-expression plasmid and the p53GFP plasmid, followed by selection with FIAU or ganciclovir.
T12507	18155-18157	PRP	denotes	We
T12509	18158-18163	RB	denotes	first
T12508	18164-18173	VBD	denotes	attempted
T12510	18174-18178	NN	denotes	RMCE
T12511	18179-18181	IN	denotes	in
T12512	18182-18186	NNS	denotes	MEFs
T12513	18187-18189	IN	denotes	by
T12514	18190-18205	VBG	denotes	electroporating
T12515	18206-18213	NN	denotes	p53RMCE
T12517	18213-18214	HYPH	denotes	/
T12518	18214-18215	SYM	denotes	−
T12516	18216-18220	NNS	denotes	MEFs
T12519	18221-18225	IN	denotes	with
T12520	18226-18227	DT	denotes	a
T12522	18228-18231	NN	denotes	Cre
T12524	18231-18232	HYPH	denotes	-
T12523	18232-18242	NN	denotes	expression
T12521	18243-18250	NN	denotes	plasmid
T12525	18251-18254	CC	denotes	and
T12526	18255-18258	DT	denotes	the
T12528	18259-18265	NN	denotes	p53GFP
T12527	18266-18273	NN	denotes	plasmid
T12529	18273-18275	,	denotes	,
T12530	18275-18283	VBN	denotes	followed
T12531	18284-18286	IN	denotes	by
T12532	18287-18296	NN	denotes	selection
T12533	18297-18301	IN	denotes	with
T12534	18302-18306	NN	denotes	FIAU
T12535	18307-18309	CC	denotes	or
T12536	18310-18321	NN	denotes	ganciclovir
T12537	18321-18322	.	denotes	.
T12538	18322-18403	sentence	denotes	Strikingly, no clones with an exchanged allele were identified (data not shown).
T12539	18323-18333	RB	denotes	Strikingly
T12541	18333-18335	,	denotes	,
T12542	18335-18337	DT	denotes	no
T12543	18338-18344	NNS	denotes	clones
T12544	18345-18349	IN	denotes	with
T12545	18350-18352	DT	denotes	an
T12547	18353-18362	VBN	denotes	exchanged
T12546	18363-18369	NN	denotes	allele
T12548	18370-18374	VBD	denotes	were
T12540	18375-18385	VBN	denotes	identified
T12549	18386-18387	-LRB-	denotes	(
T12551	18387-18391	NNS	denotes	data
T12552	18392-18395	RB	denotes	not
T12550	18396-18401	VBN	denotes	shown
T12553	18401-18402	-RRB-	denotes	)
T12554	18402-18403	.	denotes	.
T12555	18403-18606	sentence	denotes	RMCE with p53GFP in ES cells showed that p53GFP is expressed at high levels (Figure 3A), and as mentioned before, the p53 pathway that can be activated in MEFs is not readily activated in ES cells (18).
T12556	18404-18408	NN	denotes	RMCE
T12558	18409-18413	IN	denotes	with
T12559	18414-18420	NN	denotes	p53GFP
T12560	18421-18423	IN	denotes	in
T12561	18424-18426	NN	denotes	ES
T12562	18427-18432	NNS	denotes	cells
T12557	18433-18439	VBD	denotes	showed
T12563	18440-18444	IN	denotes	that
T12565	18445-18451	NN	denotes	p53GFP
T12566	18452-18454	VBZ	denotes	is
T12564	18455-18464	VBN	denotes	expressed
T12567	18465-18467	IN	denotes	at
T12568	18468-18472	JJ	denotes	high
T12569	18473-18479	NNS	denotes	levels
T12570	18480-18481	-LRB-	denotes	(
T12572	18481-18487	NN	denotes	Figure
T12571	18488-18490	NN	denotes	3A
T12573	18490-18491	-RRB-	denotes	)
T12574	18491-18493	,	denotes	,
T12575	18493-18496	CC	denotes	and
T12576	18497-18499	IN	denotes	as
T12577	18500-18509	VBN	denotes	mentioned
T12579	18510-18516	RB	denotes	before
T12580	18516-18518	,	denotes	,
T12581	18518-18521	DT	denotes	the
T12583	18522-18525	NN	denotes	p53
T12582	18526-18533	NN	denotes	pathway
T12584	18534-18538	WDT	denotes	that
T12586	18539-18542	MD	denotes	can
T12587	18543-18545	VB	denotes	be
T12585	18546-18555	VBN	denotes	activated
T12588	18556-18558	IN	denotes	in
T12589	18559-18563	NNS	denotes	MEFs
T12590	18564-18566	VBZ	denotes	is
T12591	18567-18570	RB	denotes	not
T12592	18571-18578	RB	denotes	readily
T12578	18579-18588	VBN	denotes	activated
T12593	18589-18591	IN	denotes	in
T12594	18592-18594	NN	denotes	ES
T12595	18595-18600	NNS	denotes	cells
T12596	18601-18602	-LRB-	denotes	(
T12597	18602-18604	CD	denotes	18
T12598	18604-18605	-RRB-	denotes	)
T12599	18605-18606	.	denotes	.
T12600	18606-18775	sentence	denotes	The results above suggest that high levels of p53GFP could be tolerated by ES cells but toxic to MEFs, so that MEFs in which an RMCE had occurred failed to proliferate.
T12601	18607-18610	DT	denotes	The
T12602	18611-18618	NNS	denotes	results
T12604	18619-18624	RB	denotes	above
T12603	18625-18632	VBP	denotes	suggest
T12605	18633-18637	IN	denotes	that
T12607	18638-18642	JJ	denotes	high
T12608	18643-18649	NNS	denotes	levels
T12609	18650-18652	IN	denotes	of
T12610	18653-18659	NN	denotes	p53GFP
T12611	18660-18665	MD	denotes	could
T12606	18666-18668	VB	denotes	be
T12612	18669-18678	VBN	denotes	tolerated
T12613	18679-18681	IN	denotes	by
T12614	18682-18684	NN	denotes	ES
T12615	18685-18690	NNS	denotes	cells
T12616	18691-18694	CC	denotes	but
T12617	18695-18700	JJ	denotes	toxic
T12618	18701-18703	IN	denotes	to
T12619	18704-18708	NNS	denotes	MEFs
T12620	18708-18710	,	denotes	,
T12621	18710-18712	IN	denotes	so
T12623	18713-18717	IN	denotes	that
T12624	18718-18722	NNS	denotes	MEFs
T12625	18723-18725	IN	denotes	in
T12627	18726-18731	WDT	denotes	which
T12628	18732-18734	DT	denotes	an
T12629	18735-18739	NN	denotes	RMCE
T12630	18740-18743	VBD	denotes	had
T12626	18744-18752	VBN	denotes	occurred
T12622	18753-18759	VBD	denotes	failed
T12631	18760-18762	TO	denotes	to
T12632	18763-18774	VB	denotes	proliferate
T12633	18774-18775	.	denotes	.
T12634	18775-18985	sentence	denotes	To test this possibility, p53RMCE/− MEFs were electroporated with the p53GFP replacement construct with or without a Cre-expression plasmid, then analyzed by fluorescence microscopy 48 h after electroporation.
T12635	18776-18778	TO	denotes	To
T12636	18779-18783	VB	denotes	test
T12638	18784-18788	DT	denotes	this
T12639	18789-18800	NN	denotes	possibility
T12640	18800-18802	,	denotes	,
T12641	18802-18809	NN	denotes	p53RMCE
T12643	18809-18810	HYPH	denotes	/
T12644	18810-18811	SYM	denotes	−
T12642	18812-18816	NNS	denotes	MEFs
T12645	18817-18821	VBD	denotes	were
T12637	18822-18836	VBN	denotes	electroporated
T12646	18837-18841	IN	denotes	with
T12647	18842-18845	DT	denotes	the
T12649	18846-18852	NN	denotes	p53GFP
T12650	18853-18864	NN	denotes	replacement
T12648	18865-18874	NN	denotes	construct
T12651	18875-18879	IN	denotes	with
T12652	18880-18882	CC	denotes	or
T12653	18883-18890	IN	denotes	without
T12654	18891-18892	DT	denotes	a
T12656	18893-18896	NN	denotes	Cre
T12658	18896-18897	HYPH	denotes	-
T12657	18897-18907	NN	denotes	expression
T12655	18908-18915	NN	denotes	plasmid
T12659	18915-18917	,	denotes	,
T12660	18917-18921	RB	denotes	then
T12661	18922-18930	VBN	denotes	analyzed
T12662	18931-18933	IN	denotes	by
T12663	18934-18946	NN	denotes	fluorescence
T12664	18947-18957	NN	denotes	microscopy
T12665	18958-18960	CD	denotes	48
T12666	18961-18962	NN	denotes	h
T12667	18963-18968	IN	denotes	after
T12668	18969-18984	NN	denotes	electroporation
T12669	18984-18985	.	denotes	.
T12670	18985-19203	sentence	denotes	The experiment was done without selection to enable observation of cells under conditions where a failure to proliferate would not derive from FIAU or ganciclovir toxicity but rather solely from the effects of p53GFP.
T12671	18986-18989	DT	denotes	The
T12672	18990-19000	NN	denotes	experiment
T12674	19001-19004	VBD	denotes	was
T12673	19005-19009	VBN	denotes	done
T12675	19010-19017	IN	denotes	without
T12676	19018-19027	NN	denotes	selection
T12677	19028-19030	TO	denotes	to
T12678	19031-19037	VB	denotes	enable
T12679	19038-19049	NN	denotes	observation
T12680	19050-19052	IN	denotes	of
T12681	19053-19058	NNS	denotes	cells
T12682	19059-19064	IN	denotes	under
T12683	19065-19075	NNS	denotes	conditions
T12684	19076-19081	WRB	denotes	where
T12686	19082-19083	DT	denotes	a
T12687	19084-19091	NN	denotes	failure
T12688	19092-19094	TO	denotes	to
T12689	19095-19106	VB	denotes	proliferate
T12690	19107-19112	MD	denotes	would
T12691	19113-19116	RB	denotes	not
T12685	19117-19123	VB	denotes	derive
T12692	19124-19128	IN	denotes	from
T12693	19129-19133	NN	denotes	FIAU
T12695	19134-19136	CC	denotes	or
T12696	19137-19148	NN	denotes	ganciclovir
T12694	19149-19157	NN	denotes	toxicity
T12697	19158-19161	CC	denotes	but
T12698	19162-19168	RB	denotes	rather
T12699	19169-19175	RB	denotes	solely
T12700	19176-19180	IN	denotes	from
T12701	19181-19184	DT	denotes	the
T12702	19185-19192	NNS	denotes	effects
T12703	19193-19195	IN	denotes	of
T12704	19196-19202	NN	denotes	p53GFP
T12705	19202-19203	.	denotes	.
T12706	19203-19346	sentence	denotes	We observed a few fluorescent cells only when the Cre expression plasmid was co-electroporated, suggesting that such cells resulted from RMCE.
T12707	19204-19206	PRP	denotes	We
T12708	19207-19215	VBD	denotes	observed
T12709	19216-19217	DT	denotes	a
T12711	19218-19221	JJ	denotes	few
T12712	19222-19233	JJ	denotes	fluorescent
T12710	19234-19239	NNS	denotes	cells
T12713	19240-19244	RB	denotes	only
T12714	19245-19249	WRB	denotes	when
T12716	19250-19253	DT	denotes	the
T12718	19254-19257	NN	denotes	Cre
T12719	19258-19268	NN	denotes	expression
T12717	19269-19276	NN	denotes	plasmid
T12720	19277-19280	VBD	denotes	was
T12715	19281-19298	VBN	denotes	co-electroporated
T12721	19298-19300	,	denotes	,
T12722	19300-19310	VBG	denotes	suggesting
T12723	19311-19315	IN	denotes	that
T12725	19316-19320	JJ	denotes	such
T12726	19321-19326	NNS	denotes	cells
T12724	19327-19335	VBD	denotes	resulted
T12727	19336-19340	IN	denotes	from
T12728	19341-19345	NN	denotes	RMCE
T12729	19345-19346	.	denotes	.
T12730	19346-19529	sentence	denotes	Importantly, the rare fluorescent cells had a flat, ‘fried-egg’ appearance typical of senescent cells (Figure 5A), and when plates were observed 5 days later, the cells had detached.
T12731	19347-19358	RB	denotes	Importantly
T12733	19358-19360	,	denotes	,
T12734	19360-19363	DT	denotes	the
T12736	19364-19368	JJ	denotes	rare
T12737	19369-19380	JJ	denotes	fluorescent
T12735	19381-19386	NNS	denotes	cells
T12732	19387-19390	VBD	denotes	had
T12738	19391-19392	DT	denotes	a
T12740	19393-19397	JJ	denotes	flat
T12741	19397-19399	,	denotes	,
T12742	19399-19400	``	denotes	‘
T12743	19400-19405	JJ	denotes	fried
T12745	19405-19406	HYPH	denotes	-
T12744	19406-19409	NN	denotes	egg
T12746	19409-19410	''	denotes	’
T12739	19411-19421	NN	denotes	appearance
T12747	19422-19429	JJ	denotes	typical
T12748	19430-19432	IN	denotes	of
T12749	19433-19442	JJ	denotes	senescent
T12750	19443-19448	NNS	denotes	cells
T12751	19449-19450	-LRB-	denotes	(
T12753	19450-19456	NN	denotes	Figure
T12752	19457-19459	NN	denotes	5A
T12754	19459-19460	-RRB-	denotes	)
T12755	19460-19462	,	denotes	,
T12756	19462-19465	CC	denotes	and
T12757	19466-19470	WRB	denotes	when
T12759	19471-19477	NNS	denotes	plates
T12760	19478-19482	VBD	denotes	were
T12758	19483-19491	VBN	denotes	observed
T12762	19492-19493	CD	denotes	5
T12763	19494-19498	NNS	denotes	days
T12764	19499-19504	RB	denotes	later
T12765	19504-19506	,	denotes	,
T12766	19506-19509	DT	denotes	the
T12767	19510-19515	NNS	denotes	cells
T12768	19516-19519	VBD	denotes	had
T12761	19520-19528	VBN	denotes	detached
T12769	19528-19529	.	denotes	.
T12770	19529-19659	sentence	denotes	Altogether, the results suggest that RMCE can give rise to p53GFP-expressing MEFs, but they die rapidly owing to p53GFP toxicity.
T12771	19530-19540	RB	denotes	Altogether
T12773	19540-19542	,	denotes	,
T12774	19542-19545	DT	denotes	the
T12775	19546-19553	NNS	denotes	results
T12772	19554-19561	VBP	denotes	suggest
T12776	19562-19566	IN	denotes	that
T12778	19567-19571	NN	denotes	RMCE
T12779	19572-19575	MD	denotes	can
T12777	19576-19580	VB	denotes	give
T12780	19581-19585	NN	denotes	rise
T12781	19586-19588	IN	denotes	to
T12782	19589-19595	NN	denotes	p53GFP
T12784	19595-19596	HYPH	denotes	-
T12783	19596-19606	VBG	denotes	expressing
T12785	19607-19611	NNS	denotes	MEFs
T12786	19611-19613	,	denotes	,
T12787	19613-19616	CC	denotes	but
T12788	19617-19621	PRP	denotes	they
T12789	19622-19625	VBP	denotes	die
T12790	19626-19633	RB	denotes	rapidly
T12791	19634-19639	VBG	denotes	owing
T12792	19640-19642	IN	denotes	to
T12793	19643-19649	NN	denotes	p53GFP
T12794	19650-19658	NN	denotes	toxicity
T12795	19658-19659	.	denotes	.
T12796	19659-19729	sentence	denotes	We also performed RMCE in p53RMCE/− MEFs with the p53ΔPGFP construct.
T12797	19660-19662	PRP	denotes	We
T12799	19663-19667	RB	denotes	also
T12798	19668-19677	VBD	denotes	performed
T12800	19678-19682	NN	denotes	RMCE
T12801	19683-19685	IN	denotes	in
T12802	19686-19693	NN	denotes	p53RMCE
T12804	19693-19694	HYPH	denotes	/
T12805	19694-19695	SYM	denotes	−
T12803	19696-19700	NNS	denotes	MEFs
T12806	19701-19705	IN	denotes	with
T12807	19706-19709	DT	denotes	the
T12809	19710-19718	NN	denotes	p53ΔPGFP
T12808	19719-19728	NN	denotes	construct
T12810	19728-19729	.	denotes	.
T12811	19729-19922	sentence	denotes	p53ΔPGFP-expressing MEFs were viable, recovered with an efficiency of ∼40%, and, as expected from ES cell experiments, expressed a p53ΔPGFP protein at much lower levels than p53WT (Figure 5B).
T12812	19730-19738	NN	denotes	p53ΔPGFP
T12814	19738-19739	HYPH	denotes	-
T12813	19739-19749	VBG	denotes	expressing
T12815	19750-19754	NNS	denotes	MEFs
T12816	19755-19759	VBD	denotes	were
T12817	19760-19766	JJ	denotes	viable
T12818	19766-19768	,	denotes	,
T12819	19768-19777	VBD	denotes	recovered
T12820	19778-19782	IN	denotes	with
T12821	19783-19785	DT	denotes	an
T12822	19786-19796	NN	denotes	efficiency
T12823	19797-19799	IN	denotes	of
T12824	19800-19801	SYM	denotes	∼
T12825	19801-19803	CD	denotes	40
T12826	19803-19804	NN	denotes	%
T12827	19804-19806	,	denotes	,
T12828	19806-19809	CC	denotes	and
T12829	19809-19811	,	denotes	,
T12830	19811-19813	IN	denotes	as
T12831	19814-19822	VBN	denotes	expected
T12833	19823-19827	IN	denotes	from
T12834	19828-19830	NN	denotes	ES
T12835	19831-19835	NN	denotes	cell
T12836	19836-19847	NNS	denotes	experiments
T12837	19847-19849	,	denotes	,
T12832	19849-19858	VBD	denotes	expressed
T12838	19859-19860	DT	denotes	a
T12840	19861-19869	NN	denotes	p53ΔPGFP
T12839	19870-19877	NN	denotes	protein
T12841	19878-19880	IN	denotes	at
T12842	19881-19885	RB	denotes	much
T12843	19886-19891	JJR	denotes	lower
T12844	19892-19898	NNS	denotes	levels
T12845	19899-19903	IN	denotes	than
T12846	19904-19909	NN	denotes	p53WT
T12847	19910-19911	-LRB-	denotes	(
T12849	19911-19917	NN	denotes	Figure
T12848	19918-19920	NN	denotes	5B
T12850	19920-19921	-RRB-	denotes	)
T12851	19921-19922	.	denotes	.
T12852	19922-20004	sentence	denotes	Unlike WT MEFs, p53ΔP/ΔP MEFs are unable to arrest cycling after irradiation (5).
T12853	19923-19929	IN	denotes	Unlike
T12855	19930-19932	NN	denotes	WT
T12856	19933-19937	NNS	denotes	MEFs
T12857	19937-19939	,	denotes	,
T12858	19939-19944	NN	denotes	p53ΔP
T12860	19944-19945	HYPH	denotes	/
T12859	19945-19947	NN	denotes	ΔP
T12861	19948-19952	NNS	denotes	MEFs
T12854	19953-19956	VBP	denotes	are
T12862	19957-19963	JJ	denotes	unable
T12863	19964-19966	TO	denotes	to
T12864	19967-19973	VB	denotes	arrest
T12865	19974-19981	VBG	denotes	cycling
T12866	19982-19987	IN	denotes	after
T12867	19988-19999	NN	denotes	irradiation
T12868	20000-20001	-LRB-	denotes	(
T12869	20001-20002	CD	denotes	5
T12870	20002-20003	-RRB-	denotes	)
T12871	20003-20004	.	denotes	.
T12872	20004-20191	sentence	denotes	Likewise, we found that irradiation doses that arrested p53RMCE/− MEFs (which express a wild-type p53 from the RMCE-ready locus, see Figure 2) did not arrest p53ΔPGFP/− MEFs (Figure 5C).
T12873	20005-20013	RB	denotes	Likewise
T12875	20013-20015	,	denotes	,
T12876	20015-20017	PRP	denotes	we
T12874	20018-20023	VBD	denotes	found
T12877	20024-20028	IN	denotes	that
T12879	20029-20040	NN	denotes	irradiation
T12880	20041-20046	NNS	denotes	doses
T12881	20047-20051	WDT	denotes	that
T12882	20052-20060	VBD	denotes	arrested
T12883	20061-20068	NN	denotes	p53RMCE
T12885	20068-20069	HYPH	denotes	/
T12886	20069-20070	SYM	denotes	−
T12884	20071-20075	NNS	denotes	MEFs
T12887	20076-20077	-LRB-	denotes	(
T12888	20077-20082	WDT	denotes	which
T12889	20083-20090	VBP	denotes	express
T12890	20091-20092	DT	denotes	a
T12892	20093-20097	JJ	denotes	wild
T12894	20097-20098	HYPH	denotes	-
T12893	20098-20102	NN	denotes	type
T12891	20103-20106	NN	denotes	p53
T12895	20107-20111	IN	denotes	from
T12896	20112-20115	DT	denotes	the
T12898	20116-20120	NN	denotes	RMCE
T12900	20120-20121	HYPH	denotes	-
T12899	20121-20126	JJ	denotes	ready
T12897	20127-20132	NN	denotes	locus
T12901	20132-20134	,	denotes	,
T12902	20134-20137	VBP	denotes	see
T12903	20138-20144	NN	denotes	Figure
T12904	20145-20146	CD	denotes	2
T12905	20146-20147	-RRB-	denotes	)
T12906	20148-20151	VBD	denotes	did
T12907	20152-20155	RB	denotes	not
T12878	20156-20162	VB	denotes	arrest
T12908	20163-20171	NN	denotes	p53ΔPGFP
T12910	20171-20172	HYPH	denotes	/
T12911	20172-20173	SYM	denotes	−
T12909	20174-20178	NNS	denotes	MEFs
T12912	20179-20180	-LRB-	denotes	(
T12914	20180-20186	NN	denotes	Figure
T12913	20187-20189	NN	denotes	5C
T12915	20189-20190	-RRB-	denotes	)
T12916	20190-20191	.	denotes	.
T12917	20191-20305	sentence	denotes	These data show that a single RMCE-ASAP reaction in heterozygous MEFs enables detection of a recessive phenotype.
T12918	20192-20197	DT	denotes	These
T12919	20198-20202	NNS	denotes	data
T12920	20203-20207	VBP	denotes	show
T12921	20208-20212	IN	denotes	that
T12923	20213-20214	DT	denotes	a
T12925	20215-20221	JJ	denotes	single
T12926	20222-20226	NN	denotes	RMCE
T12928	20226-20227	HYPH	denotes	-
T12927	20227-20231	NN	denotes	ASAP
T12924	20232-20240	NN	denotes	reaction
T12929	20241-20243	IN	denotes	in
T12930	20244-20256	JJ	denotes	heterozygous
T12931	20257-20261	NNS	denotes	MEFs
T12922	20262-20269	VBZ	denotes	enables
T12932	20270-20279	NN	denotes	detection
T12933	20280-20282	IN	denotes	of
T12934	20283-20284	DT	denotes	a
T12936	20285-20294	JJ	denotes	recessive
T12935	20295-20304	NN	denotes	phenotype
T12937	20304-20305	.	denotes	.
T12938	20305-20508	sentence	denotes	The results confirm that deleting the proline rich domain leads to less active p53 with impaired cell cycle control, and also indicate that a GFP C-terminal fusion can dramatically alter p53 regulation.
T12939	20306-20309	DT	denotes	The
T12940	20310-20317	NNS	denotes	results
T12941	20318-20325	VBP	denotes	confirm
T12942	20326-20330	IN	denotes	that
T12944	20331-20339	VBG	denotes	deleting
T12945	20340-20343	DT	denotes	the
T12947	20344-20351	NN	denotes	proline
T12948	20352-20356	JJ	denotes	rich
T12946	20357-20363	NN	denotes	domain
T12943	20364-20369	VBZ	denotes	leads
T12949	20370-20372	IN	denotes	to
T12950	20373-20377	RBR	denotes	less
T12951	20378-20384	JJ	denotes	active
T12952	20385-20388	NN	denotes	p53
T12953	20389-20393	IN	denotes	with
T12954	20394-20402	JJ	denotes	impaired
T12956	20403-20407	NN	denotes	cell
T12957	20408-20413	NN	denotes	cycle
T12955	20414-20421	NN	denotes	control
T12958	20421-20423	,	denotes	,
T12959	20423-20426	CC	denotes	and
T12960	20427-20431	RB	denotes	also
T12961	20432-20440	VBP	denotes	indicate
T12962	20441-20445	IN	denotes	that
T12964	20446-20447	DT	denotes	a
T12966	20448-20451	NN	denotes	GFP
T12967	20452-20453	NN	denotes	C
T12968	20453-20454	HYPH	denotes	-
T12969	20454-20462	JJ	denotes	terminal
T12965	20463-20469	NN	denotes	fusion
T12970	20470-20473	MD	denotes	can
T12971	20474-20486	RB	denotes	dramatically
T12963	20487-20492	VB	denotes	alter
T12972	20493-20496	NN	denotes	p53
T12973	20497-20507	NN	denotes	regulation
T12974	20507-20508	.	denotes	.
T12975	20508-20558	sentence	denotes	A summary of our results is presented in Table 1.
T12976	20509-20510	DT	denotes	A
T12977	20511-20518	NN	denotes	summary
T12979	20519-20521	IN	denotes	of
T12980	20522-20525	PRP$	denotes	our
T12981	20526-20533	NNS	denotes	results
T12982	20534-20536	VBZ	denotes	is
T12978	20537-20546	VBN	denotes	presented
T12983	20547-20549	IN	denotes	in
T12984	20550-20555	NN	denotes	Table
T12985	20556-20557	CD	denotes	1
T12986	20557-20558	.	denotes	.
T12987	20558-20772	sentence	denotes	These data report the development and implementation of an improved RMCE approach that enables efficient allele modification in ES cells to generate mice and in heterozygous MEFs to accelerate phenotypic analyses.
T12988	20559-20564	DT	denotes	These
T12989	20565-20569	NNS	denotes	data
T12990	20570-20576	VBP	denotes	report
T12991	20577-20580	DT	denotes	the
T12992	20581-20592	NN	denotes	development
T12993	20593-20596	CC	denotes	and
T12994	20597-20611	NN	denotes	implementation
T12995	20612-20614	IN	denotes	of
T12996	20615-20617	DT	denotes	an
T12998	20618-20626	VBN	denotes	improved
T12999	20627-20631	NN	denotes	RMCE
T12997	20632-20640	NN	denotes	approach
T13000	20641-20645	WDT	denotes	that
T13001	20646-20653	VBZ	denotes	enables
T13002	20654-20663	JJ	denotes	efficient
T13004	20664-20670	NN	denotes	allele
T13003	20671-20683	NN	denotes	modification
T13005	20684-20686	IN	denotes	in
T13006	20687-20689	NN	denotes	ES
T13007	20690-20695	NNS	denotes	cells
T13008	20696-20698	TO	denotes	to
T13009	20699-20707	VB	denotes	generate
T13010	20708-20712	NNS	denotes	mice
T13011	20713-20716	CC	denotes	and
T13012	20717-20719	IN	denotes	in
T13014	20720-20732	JJ	denotes	heterozygous
T13015	20733-20737	NNS	denotes	MEFs
T13016	20738-20740	TO	denotes	to
T13013	20741-20751	VB	denotes	accelerate
T13017	20752-20762	JJ	denotes	phenotypic
T13018	20763-20771	NNS	denotes	analyses
T13019	20771-20772	.	denotes	.
T13020	20772-21035	sentence	denotes	The success of RMCE-ASAP relied on the integrated use of inverted heterologous loxP sites, a positive/negative selection marker that preserves the germline capacity of ES cells, and, for somatic cells, the existence of a knock-out allele of the gene of interest.
T13021	20773-20776	DT	denotes	The
T13022	20777-20784	NN	denotes	success
T13024	20785-20787	IN	denotes	of
T13025	20788-20792	NN	denotes	RMCE
T13027	20792-20793	HYPH	denotes	-
T13026	20793-20797	NN	denotes	ASAP
T13023	20798-20804	VBN	denotes	relied
T13028	20805-20807	IN	denotes	on
T13029	20808-20811	DT	denotes	the
T13031	20812-20822	VBN	denotes	integrated
T13030	20823-20826	NN	denotes	use
T13032	20827-20829	IN	denotes	of
T13033	20830-20838	VBN	denotes	inverted
T13035	20839-20851	JJ	denotes	heterologous
T13036	20852-20856	NN	denotes	loxP
T13034	20857-20862	NNS	denotes	sites
T13037	20862-20864	,	denotes	,
T13038	20864-20865	DT	denotes	a
T13040	20866-20874	JJ	denotes	positive
T13042	20874-20875	HYPH	denotes	/
T13041	20875-20883	JJ	denotes	negative
T13043	20884-20893	NN	denotes	selection
T13039	20894-20900	NN	denotes	marker
T13044	20901-20905	WDT	denotes	that
T13045	20906-20915	VBZ	denotes	preserves
T13046	20916-20919	DT	denotes	the
T13048	20920-20928	NN	denotes	germline
T13047	20929-20937	NN	denotes	capacity
T13049	20938-20940	IN	denotes	of
T13050	20941-20943	NN	denotes	ES
T13051	20944-20949	NNS	denotes	cells
T13052	20949-20951	,	denotes	,
T13053	20951-20954	CC	denotes	and
T13054	20954-20956	,	denotes	,
T13055	20956-20959	IN	denotes	for
T13057	20960-20967	JJ	denotes	somatic
T13058	20968-20973	NNS	denotes	cells
T13059	20973-20975	,	denotes	,
T13060	20975-20978	DT	denotes	the
T13056	20979-20988	NN	denotes	existence
T13061	20989-20991	IN	denotes	of
T13062	20992-20993	DT	denotes	a
T13064	20994-20999	VB	denotes	knock
T13065	20999-21000	HYPH	denotes	-
T13066	21000-21003	RP	denotes	out
T13063	21004-21010	NN	denotes	allele
T13067	21011-21013	IN	denotes	of
T13068	21014-21017	DT	denotes	the
T13069	21018-21022	NN	denotes	gene
T13070	21023-21025	IN	denotes	of
T13071	21026-21034	NN	denotes	interest
T13072	21034-21035	.	denotes	.
T13073	21035-21222	sentence	denotes	These characteristics should make RMCE-ASAP a robust and general technology for analysis of mammalian genes under conditions that preserve normal control mechanisms in different tissues.
T13074	21036-21041	DT	denotes	These
T13075	21042-21057	NNS	denotes	characteristics
T13077	21058-21064	MD	denotes	should
T13076	21065-21069	VB	denotes	make
T13078	21070-21074	NN	denotes	RMCE
T13080	21074-21075	HYPH	denotes	-
T13079	21075-21079	NN	denotes	ASAP
T13082	21080-21081	DT	denotes	a
T13083	21082-21088	JJ	denotes	robust
T13084	21089-21092	CC	denotes	and
T13085	21093-21100	JJ	denotes	general
T13081	21101-21111	NN	denotes	technology
T13086	21112-21115	IN	denotes	for
T13087	21116-21124	NN	denotes	analysis
T13088	21125-21127	IN	denotes	of
T13089	21128-21137	JJ	denotes	mammalian
T13090	21138-21143	NNS	denotes	genes
T13091	21144-21149	IN	denotes	under
T13092	21150-21160	NNS	denotes	conditions
T13093	21161-21165	WDT	denotes	that
T13094	21166-21174	VBP	denotes	preserve
T13095	21175-21181	JJ	denotes	normal
T13097	21182-21189	NN	denotes	control
T13096	21190-21200	NNS	denotes	mechanisms
T13098	21201-21203	IN	denotes	in
T13099	21204-21213	JJ	denotes	different
T13100	21214-21221	NNS	denotes	tissues
T13101	21221-21222	.	denotes	.
T13102	21222-21382	sentence	denotes	In addition, RMCE-ASAP could be used to generate fibroblastic cell lines tailored for the repeated targeting of widely studied genes (p53, c-myc, NF-KB, etc.).
T13103	21223-21225	IN	denotes	In
T13105	21226-21234	NN	denotes	addition
T13106	21234-21236	,	denotes	,
T13107	21236-21240	NN	denotes	RMCE
T13109	21240-21241	HYPH	denotes	-
T13108	21241-21245	NN	denotes	ASAP
T13110	21246-21251	MD	denotes	could
T13111	21252-21254	VB	denotes	be
T13104	21255-21259	VBN	denotes	used
T13112	21260-21262	TO	denotes	to
T13113	21263-21271	VB	denotes	generate
T13114	21272-21284	JJ	denotes	fibroblastic
T13116	21285-21289	NN	denotes	cell
T13115	21290-21295	NNS	denotes	lines
T13117	21296-21304	VBN	denotes	tailored
T13118	21305-21308	IN	denotes	for
T13119	21309-21312	DT	denotes	the
T13121	21313-21321	VBN	denotes	repeated
T13120	21322-21331	NN	denotes	targeting
T13122	21332-21334	IN	denotes	of
T13123	21335-21341	RB	denotes	widely
T13124	21342-21349	VBN	denotes	studied
T13125	21350-21355	NNS	denotes	genes
T13126	21356-21357	-LRB-	denotes	(
T13127	21357-21360	NN	denotes	p53
T13128	21360-21362	,	denotes	,
T13129	21362-21363	NN	denotes	c
T13131	21363-21364	HYPH	denotes	-
T13130	21364-21367	NN	denotes	myc
T13132	21367-21369	,	denotes	,
T13133	21369-21371	NN	denotes	NF
T13135	21371-21372	HYPH	denotes	-
T13134	21372-21374	NN	denotes	KB
T13136	21374-21376	,	denotes	,
T13137	21376-21380	FW	denotes	etc.
T13138	21380-21381	-RRB-	denotes	)
T13139	21381-21382	.	denotes	.
R10	T448	T447	amod	targeting,gene
R100	T549	T550	det	the,approach
R1000	T3385	T3381	compound	selection,marker
R1001	T3386	T3387	dep	that,preserves
R1002	T3387	T3381	relcl	preserves,marker
R1003	T3388	T3389	det	the,capacity
R1004	T3389	T3387	dobj	capacity,preserves
R1005	T3390	T3389	compound	germline,capacity
R1006	T3391	T3389	prep	of,capacity
R1007	T3392	T3393	compound	ES,cells
R1008	T3393	T3391	pobj	cells,of
R1009	T3394	T3381	punct	", ",marker
R101	T550	T545	nsubjpass	approach,Adapted
R1010	T3395	T3381	cc	and,marker
R1011	T3396	T3397	det	the,power
R1012	T3397	T3381	conj	power,marker
R1013	T3398	T3397	prep	of,power
R1014	T3399	T3400	compound	mouse,genetics
R1015	T3400	T3398	pobj	genetics,of
R1016	T3401	T3402	aux	to,expedite
R1017	T3402	T3367	advcl	expedite,relies
R1018	T3403	T3404	amod	phenotypic,analyses
R1019	T3404	T3402	dobj	analyses,expedite
R102	T551	T545	auxpass	was,Adapted
R1020	T3405	T3367	punct	.,relies
R1021	T3407	T3408	nsubj	We,show
R1022	T3409	T3410	mark	that,enables
R1023	T3410	T3408	ccomp	enables,show
R1024	T3411	T3412	poss	our,approach
R1025	T3412	T3410	nsubj	approach,enables
R1026	T3413	T3414	amod	efficient,targeting
R1027	T3414	T3410	dobj	targeting,enables
R1028	T3415	T3414	prep	of,targeting
R1029	T3416	T3417	amod	marker,free
R103	T552	T545	prep	for,Adapted
R1030	T3417	T3419	amod	free,mutations
R1031	T3418	T3417	punct	-,free
R1032	T3419	T3415	pobj	mutations,of
R1033	T3420	T3419	prep	at,mutations
R1034	T3421	T3422	det	the,locus
R1035	T3422	T3420	pobj	locus,at
R1036	T3423	T3422	compound	p53,locus
R1037	T3424	T3414	prep	in,targeting
R1038	T3425	T3426	compound	ES,cells
R1039	T3426	T3424	pobj	cells,in
R104	T553	T552	pobj	targeting,for
R1040	T3427	T3428	aux	to,generate
R1041	T3428	T3410	xcomp	generate,enables
R1042	T3429	T3430	amod	mutant,mice
R1043	T3430	T3428	dobj	mice,generate
R1044	T3431	T3408	punct	", ",show
R1045	T3432	T3408	cc	but,show
R1046	T3433	T3434	advmod	more,importantly
R1047	T3434	T3435	advmod	importantly,Adapted
R1048	T3435	T3408	conj	Adapted,show
R1049	T3436	T3435	punct	", ",Adapted
R105	T554	T545	prep	in,Adapted
R1050	T4611	T4612	compound	Targeting,construct
R1051	T4613	T4612	prep	for,construct
R1052	T4614	T4615	det	a,locus
R1053	T4615	T4613	pobj	locus,for
R1054	T4616	T4615	nmod	p53,locus
R1055	T4617	T4618	npadvmod	RMCE,ready
R1056	T4618	T4615	amod	ready,locus
R1057	T4619	T4618	punct	-,ready
R1058	T4621	T4622	nsubj	Details,are
R1059	T4623	T4621	prep	for,Details
R106	T555	T556	amod	Somatic,cells
R1060	T4624	T4625	compound	plasmid,construction
R1061	T4625	T4623	pobj	construction,for
R1062	T4626	T4622	acomp	available,are
R1063	T4627	T4622	prep	upon,are
R1064	T4628	T4627	pobj	request,upon
R1065	T4629	T4622	prep	owing,are
R1066	T4630	T4629	prep	to,owing
R1067	T4631	T4632	compound	space,limitations
R1068	T4632	T4630	pobj	limitations,to
R1069	T4633	T4632	acl	mandating,limitations
R107	T556	T554	pobj	cells,in
R1070	T4634	T4635	det	a,outline
R1071	T4635	T4633	dobj	outline,mandating
R1072	T4636	T4635	amod	brief,outline
R1073	T4637	T4622	punct	.,are
R1074	T4639	T4640	nsubj	We,started
R1075	T4641	T4640	prep	from,started
R1076	T4642	T4643	det	a,1L
R1077	T4643	T4641	pobj	1L,from
R1078	T4644	T4643	compound	plasmid,1L
R1079	T4645	T4643	compound	L3,1L
R108	T557	T558	aux	to,Accelerate
R1080	T4646	T4643	punct	-,1L
R1081	T4647	T4643	acl	containing,1L
R1082	T4648	T4649	amod	heterologous,sites
R1083	T4649	T4647	dobj	sites,containing
R1084	T4650	T4649	compound	loxP,sites
R1085	T4651	T4652	punct	(,is
R1086	T4652	T4640	parataxis	is,started
R1087	T4653	T4654	nsubj	L3,is
R1088	T4654	T4652	ccomp	is,is
R1089	T4655	T4656	det	the,loxP257
R109	T558	T545	advcl	Accelerate,Adapted
R1090	T4656	T4654	attr	loxP257,is
R1091	T4657	T4656	amod	mutant,loxP257
R1092	T4658	T4659	advmod	recently,described
R1093	T4659	T4656	acl	described,loxP257
R1094	T4660	T4661	punct	(,14
R1095	T4661	T4654	parataxis	14,is
R1096	T4662	T4661	punct	),14
R1097	T4663	T4652	punct	", ",is
R1098	T4664	T4652	nsubj	1L,is
R1099	T4665	T4666	det	an,loxP
R11	T449	T446	prep	for,method
R110	T559	T558	dobj	Phenotyping,Accelerate
R1100	T4666	T4652	attr	loxP,is
R1101	T4667	T4666	amod	inverted,loxP
R1102	T4668	T4666	compound	WT,loxP
R1103	T4669	T4652	punct	),is
R1104	T4670	T4640	punct	.,started
R1105	T4672	T4673	det	The,loxP
R1106	T4673	T4675	nsubj	loxP,differ
R1107	T4674	T4673	compound	WT,loxP
R1108	T4675	T4678	ccomp	differ,is
R1109	T4676	T4673	cc	and,loxP
R111	T560	T559	punct	(,Phenotyping
R1110	T4677	T4673	conj	loxP257,loxP
R1111	T4679	T4675	prep	in,differ
R1112	T4680	T4681	poss	their,sequences
R1113	T4681	T4679	pobj	sequences,in
R1114	T4682	T4681	compound	spacer,sequences
R1115	T4683	T4678	punct	: ,is
R1116	T4684	T4685	det	the,sequence
R1117	T4685	T4678	nsubj	sequence,is
R1118	T4686	T4685	compound	spacer,sequence
R1119	T4687	T4678	attr	5,is
R112	T561	T562	compound	RMCE,ASAP
R1120	T4688	T4687	punct	′,5
R1121	T4689	T4687	punct	-,5
R1122	T4690	T4687	amod	ATGTATGC,5
R1123	T4691	T4687	punct	-,5
R1124	T4692	T4687	nummod	3,5
R1125	T4693	T4687	punct	′,5
R1126	T4694	T4687	prep	for,5
R1127	T4695	T4696	compound	WT,loxP
R1128	T4696	T4694	pobj	loxP,for
R1129	T4697	T4687	cc	and,5
R113	T562	T559	appos	ASAP,Phenotyping
R1130	T4698	T4699	nummod	5,AAGTCTCC
R1131	T4699	T4687	conj	AAGTCTCC,5
R1132	T4700	T4698	punct	′,5
R1133	T4701	T4699	punct	-,AAGTCTCC
R1134	T4702	T4699	punct	-,AAGTCTCC
R1135	T4703	T4699	nummod	3,AAGTCTCC
R1136	T4704	T4699	punct	′,AAGTCTCC
R1137	T4705	T4699	prep	for,AAGTCTCC
R1138	T4706	T4705	pobj	loxP257,for
R1139	T4707	T4678	punct	.,is
R114	T563	T562	punct	-,ASAP
R1140	T4709	T4710	det	The,mutations
R1141	T4710	T4712	nsubj	mutations,prevent
R1142	T4711	T4710	nummod	three,mutations
R1143	T4712	T4718	ccomp	prevent,occurred
R1144	T4713	T4710	prep	in,mutations
R1145	T4714	T4715	det	the,sequence
R1146	T4715	T4713	pobj	sequence,in
R1147	T4716	T4715	compound	loxP257,sequence
R1148	T4717	T4715	compound	spacer,sequence
R1149	T4719	T4720	nsubj	it,recombine
R115	T564	T545	punct	),Adapted
R1150	T4720	T4712	ccomp	recombine,prevent
R1151	T4721	T4720	aux	to,recombine
R1152	T4722	T4720	prep	with,recombine
R1153	T4723	T4724	compound	WT,loxP
R1154	T4724	T4722	pobj	loxP,with
R1155	T4725	T4712	punct	", ",prevent
R1156	T4726	T4712	advcl	ensuring,prevent
R1157	T4727	T4728	amod	efficient,RMCE
R1158	T4728	T4726	dobj	RMCE,ensuring
R1159	T4729	T4726	prep	in,ensuring
R116	T565	T545	punct	.,Adapted
R1160	T4730	T4731	amod	several,lines
R1161	T4731	T4729	pobj	lines,in
R1162	T4732	T4731	compound	cell,lines
R1163	T4733	T4718	punct	: ,occurred
R1164	T4734	T4735	amod	accurate,RMCE
R1165	T4735	T4718	nsubj	RMCE,occurred
R1166	T4736	T4735	prep	with,RMCE
R1167	T4737	T4738	det	these,sites
R1168	T4738	T4736	pobj	sites,with
R1169	T4739	T4738	compound	loxP,sites
R117	T567	T568	nsubj	We,provide
R1170	T4740	T4718	prep	with,occurred
R1171	T4741	T4742	det	an,frequency
R1172	T4742	T4740	pobj	frequency,with
R1173	T4743	T4742	amod	average,frequency
R1174	T4744	T4742	prep	of,frequency
R1175	T4745	T4746	nummod	81,%
R1176	T4746	T4744	pobj	%,of
R1177	T4747	T4742	prep	at,frequency
R1178	T4748	T4749	nummod	two,loci
R1179	T4749	T4747	pobj	loci,at
R118	T569	T568	dobj	proof,provide
R1180	T4750	T4749	prep	in,loci
R1181	T4751	T4752	compound	CHO,cells
R1182	T4752	T4750	pobj	cells,in
R1183	T4753	T4742	cc	and,frequency
R1184	T4754	T4755	det	an,frequency
R1185	T4755	T4742	conj	frequency,frequency
R1186	T4756	T4755	amod	average,frequency
R1187	T4757	T4755	prep	of,frequency
R1188	T4758	T4759	nummod	69,%
R1189	T4759	T4757	pobj	%,of
R119	T570	T569	punct	-,proof
R1190	T4760	T4755	prep	at,frequency
R1191	T4761	T4762	nummod	four,loci
R1192	T4762	T4760	pobj	loci,at
R1193	T4763	T4762	prep	in,loci
R1194	T4764	T4765	compound	Hela,cells
R1195	T4765	T4763	pobj	cells,in
R1196	T4766	T4767	punct	(,14
R1197	T4767	T4718	parataxis	14,occurred
R1198	T4768	T4767	punct	),14
R1199	T4769	T4718	punct	.,occurred
R12	T450	T451	nmod	ES,cells
R120	T571	T569	prep	of,proof
R1200	T4771	T4772	det	The,plasmid
R1201	T4772	T4776	nsubjpass	plasmid,modified
R1202	T4773	T4774	compound	L3,1L
R1203	T4774	T4772	compound	1L,plasmid
R1204	T4775	T4774	punct	-,1L
R1205	T4777	T4776	auxpass	was,modified
R1206	T4778	T4776	advmod	first,modified
R1207	T4779	T4780	aux	to,include
R1208	T4780	T4776	advcl	include,modified
R1209	T4781	T4782	det	a,ClaI
R121	T572	T571	punct	-,of
R1210	T4782	T4780	dobj	ClaI,include
R1211	T4783	T4782	cc	and,ClaI
R1212	T4784	T4785	det	a,site
R1213	T4785	T4782	conj	site,ClaI
R1214	T4786	T4785	compound	FseI,site
R1215	T4787	T4780	prep	between,include
R1216	T4788	T4789	det	the,sites
R1217	T4789	T4787	pobj	sites,between
R1218	T4790	T4789	compound	LoxP,sites
R1219	T4791	T4776	punct	", ",modified
R122	T573	T571	pobj	concept,of
R1220	T4792	T4776	advcl	leading,modified
R1221	T4793	T4792	prep	to,leading
R1222	T4794	T4795	compound	plasmid,1L
R1223	T4795	T4793	pobj	1L,to
R1224	T4796	T4795	compound	L3,1L
R1225	T4797	T4795	punct	-,1L
R1226	T4798	T4795	compound	CF,1L
R1227	T4799	T4795	punct	-,1L
R1228	T4800	T4776	punct	.,modified
R1229	T4802	T4803	nsubj	We,modified
R123	T574	T568	prep	for,provide
R1230	T4804	T4803	advmod	next,modified
R1231	T4805	T4806	det	a,plasmid
R1232	T4806	T4803	dobj	plasmid,modified
R1233	T4807	T4806	compound	puroΔTK,plasmid
R1234	T4808	T4806	punct	(,plasmid
R1235	T4809	T4806	appos	YTC37,plasmid
R1236	T4810	T4809	punct	", ",YTC37
R1237	T4811	T4812	det	a,gift
R1238	T4812	T4809	appos	gift,YTC37
R1239	T4813	T4812	compound	kind,gift
R124	T575	T574	pobj	this,for
R1240	T4814	T4812	prep	from,gift
R1241	T4815	T4816	compound	A.,Bradley
R1242	T4816	T4814	pobj	Bradley,from
R1243	T4817	T4803	punct	),modified
R1244	T4818	T4803	prep	by,modified
R1245	T4819	T4818	pcomp	using,by
R1246	T4820	T4819	dobj	oligonucleotides,using
R1247	T4821	T4822	aux	to,destroy
R1248	T4822	T4819	advcl	destroy,using
R1249	T4823	T4824	det	a,site
R125	T576	T568	prep	at,provide
R1250	T4824	T4822	dobj	site,destroy
R1251	T4825	T4824	compound	NotI,site
R1252	T4826	T4824	advmod	downstream,site
R1253	T4827	T4826	prep	of,downstream
R1254	T4828	T4829	det	the,gene
R1255	T4829	T4827	pobj	gene,of
R1256	T4830	T4829	compound	puroΔTK,gene
R1257	T4831	T4822	cc	and,destroy
R1258	T4832	T4822	conj	introduce,destroy
R1259	T4833	T4834	det	a,site
R126	T577	T578	det	the,locus
R1260	T4834	T4832	dobj	site,introduce
R1261	T4835	T4834	compound	NotI,site
R1262	T4836	T4832	advmod	upstream,introduce
R1263	T4837	T4832	punct	", ",introduce
R1264	T4838	T4832	cc	and,introduce
R1265	T4839	T4840	det	a,site
R1266	T4840	T4832	conj	site,introduce
R1267	T4841	T4840	compound	FseI,site
R1268	T4842	T4840	advmod	downstream,site
R1269	T4843	T4842	prep	of,downstream
R127	T578	T576	pobj	locus,at
R1270	T4844	T4845	det	the,gene
R1271	T4845	T4843	pobj	gene,of
R1272	T4846	T4847	punct	(,leading
R1273	T4847	T4840	parataxis	leading,site
R1274	T4848	T4847	prep	to,leading
R1275	T4849	T4850	compound	plasmid,F
R1276	T4850	T4848	pobj	F,to
R1277	T4851	T4850	compound	CN,F
R1278	T4852	T4850	punct	-,F
R1279	T4853	T4850	compound	PuroΔTK,F
R128	T579	T578	compound	p53,locus
R1280	T4854	T4850	punct	-,F
R1281	T4855	T4847	punct	),leading
R1282	T4856	T4803	punct	.,modified
R1283	T4858	T4859	advmod	Next,subcloned
R1284	T4860	T4859	punct	", ",subcloned
R1285	T4861	T4862	det	a,fragment
R1286	T4862	T4859	nsubjpass	fragment,subcloned
R1287	T4863	T4864	nmod	PmlI,MfeI
R1288	T4864	T4862	nmod	MfeI,fragment
R1289	T4865	T4864	punct	-,MfeI
R129	T580	T568	punct	", ",provide
R1290	T4866	T4867	nummod	6.3,kb
R1291	T4867	T4862	compound	kb,fragment
R1292	T4868	T4862	prep	from,fragment
R1293	T4869	T4868	pobj	Trp53,from
R1294	T4870	T4859	auxpass	was,subcloned
R1295	T4871	T4859	prep	in,subcloned
R1296	T4872	T4873	det	a,KSII+
R1297	T4873	T4871	pobj	KSII+,in
R1298	T4874	T4873	amod	modified,KSII+
R1299	T4875	T4873	compound	pBluescript,KSII+
R13	T451	T449	pobj	cells,for
R130	T581	T568	prep	by,provide
R1300	T4876	T4877	punct	(,pBS
R1301	T4877	T4873	parataxis	pBS,KSII+
R1302	T4878	T4877	punct	", ",pBS
R1303	T4879	T4877	npadvmod	Stratagene,pBS
R1304	T4880	T4877	punct	),pBS
R1305	T4881	T4859	punct	", ",subcloned
R1306	T4882	T4859	cc	and,subcloned
R1307	T4883	T4884	det	the,plasmid
R1308	T4884	T4886	nsubjpass	plasmid,digested
R1309	T4885	T4884	amod	resulting,plasmid
R131	T582	T581	pcomp	showing,by
R1310	T4886	T4859	conj	digested,subcloned
R1311	T4887	T4886	auxpass	was,digested
R1312	T4888	T4886	prep	with,digested
R1313	T4889	T4888	pobj	SwaI,with
R1314	T4890	T4891	aux	to,introduce
R1315	T4891	T4886	advcl	introduce,digested
R1316	T4892	T4893	det	an,site
R1317	T4893	T4891	dobj	site,introduce
R1318	T4894	T4893	compound	EagI,site
R1319	T4895	T4891	punct	", ",introduce
R132	T583	T584	amod	efficient,targeting
R1320	T4896	T4891	advcl	leading,introduce
R1321	T4897	T4896	prep	to,leading
R1322	T4898	T4897	pobj	p53PmlEag,to
R1323	T4899	T4898	punct	", ",p53PmlEag
R1324	T4900	T4901	det	a,plasmid
R1325	T4901	T4898	appos	plasmid,p53PmlEag
R1326	T4902	T4901	acl	containing,plasmid
R1327	T4903	T4904	nmod	exons,11
R1328	T4904	T4902	dobj	11,containing
R1329	T4905	T4904	nummod	2,11
R133	T584	T582	dobj	targeting,showing
R1330	T4906	T4904	punct	–,11
R1331	T4907	T4904	prep	of,11
R1332	T4908	T4907	pobj	p53,of
R1333	T4909	T4886	punct	.,digested
R1334	T4911	T4912	nsubj	We,inserted
R1335	T4913	T4912	advmod	then,inserted
R1336	T4914	T4915	det	a,fragment
R1337	T4915	T4912	dobj	fragment,inserted
R1338	T4916	T4917	nummod	5.5,kb
R1339	T4917	T4915	compound	kb,fragment
R134	T585	T584	prep	in,targeting
R1340	T4918	T4919	compound	ClaI,EagI
R1341	T4919	T4915	compound	EagI,fragment
R1342	T4920	T4919	punct	-,EagI
R1343	T4921	T4915	prep	from,fragment
R1344	T4922	T4921	pobj	p53PmlEag,from
R1345	T4923	T4915	prep	in,fragment
R1346	T4924	T4925	compound	plasmid,F
R1347	T4925	T4923	pobj	F,in
R1348	T4926	T4925	compound	CN,F
R1349	T4927	T4925	punct	-,F
R135	T586	T585	pobj	fibroblasts,in
R1350	T4928	T4925	compound	PuroΔTK,F
R1351	T4929	T4925	punct	-,F
R1352	T4930	T4915	acl	digested,fragment
R1353	T4931	T4930	agent	by,digested
R1354	T4932	T4931	pobj	ClaI,by
R1355	T4933	T4932	cc	and,ClaI
R1356	T4934	T4932	conj	NotI,ClaI
R1357	T4935	T4912	punct	", ",inserted
R1358	T4936	T4912	cc	and,inserted
R1359	T4937	T4912	conj	inserted,inserted
R136	T587	T584	punct	", ",targeting
R1360	T4938	T4939	det	the,fragment
R1361	T4939	T4937	dobj	fragment,inserted
R1362	T4940	T4939	amod	resulting,fragment
R1363	T4941	T4937	prep	between,inserted
R1364	T4942	T4943	det	the,sites
R1365	T4943	T4941	pobj	sites,between
R1366	T4944	T4943	compound	loxP,sites
R1367	T4945	T4943	prep	of,sites
R1368	T4946	T4947	compound	L3,1L
R1369	T4947	T4945	pobj	1L,of
R137	T588	T584	cc	and,targeting
R1370	T4948	T4947	punct	-,1L
R1371	T4949	T4947	compound	CF,1L
R1372	T4950	T4947	punct	-,1L
R1373	T4951	T4937	prep	by,inserted
R1374	T4952	T4953	nmod	ClaI,digestion
R1375	T4953	T4951	pobj	digestion,by
R1376	T4954	T4952	cc	and,ClaI
R1377	T4955	T4952	conj	FseI,ClaI
R1378	T4956	T4937	punct	", ",inserted
R1379	T4957	T4937	advcl	leading,inserted
R138	T589	T590	amod	rapid,out
R1380	T4958	T4957	prep	to,leading
R1381	T4959	T4960	compound	L3,1L
R1382	T4960	T4958	pobj	1L,to
R1383	T4961	T4960	punct	-,1L
R1384	T4962	T4960	compound	p53PmlEagPuroΔTK,1L
R1385	T4963	T4960	punct	-,1L
R1386	T4964	T4912	punct	.,inserted
R1387	T4966	T4967	nsubj	We,engineered
R1388	T4967	T4969	ccomp	engineered,performed
R1389	T4968	T4967	advmod	next,engineered
R139	T590	T584	conj	out,targeting
R1390	T4970	T4971	det	a,plasmid
R1391	T4971	T4967	dobj	plasmid,engineered
R1392	T4972	T4971	acl	containing,plasmid
R1393	T4973	T4974	det	the,region
R1394	T4974	T4972	dobj	region,containing
R1395	T4975	T4974	prep	for,region
R1396	T4976	T4975	pobj	3,for
R1397	T4977	T4976	punct	′,3
R1398	T4978	T4976	punct	-,3
R1399	T4979	T4976	amod	homology,3
R14	T452	T450	cc	and,ES
R140	T591	T590	amod	phenotypic,out
R1400	T4980	T4972	advmod	downstream,containing
R1401	T4981	T4980	prep	of,downstream
R1402	T4982	T4983	det	the,gene
R1403	T4983	T4981	pobj	gene,of
R1404	T4984	T4983	compound	p53,gene
R1405	T4985	T4983	cc	and,gene
R1406	T4986	T4987	det	the,gene
R1407	T4987	T4983	conj	gene,gene
R1408	T4988	T4987	compound	DTA,gene
R1409	T4989	T4967	prep	in,engineered
R141	T592	T590	compound	read,out
R1410	T4990	T4991	nummod	two,steps
R1411	T4991	T4989	pobj	steps,in
R1412	T4992	T4969	punct	: ,performed
R1413	T4993	T4994	punct	(,i
R1414	T4994	T4969	meta	i,performed
R1415	T4995	T4994	punct	),i
R1416	T4996	T4969	nsubj	we,performed
R1417	T4997	T4998	det	a,ligation
R1418	T4998	T4969	dobj	ligation,performed
R1419	T4999	T5000	nummod	three,way
R142	T593	T590	punct	-,out
R1420	T5000	T4998	compound	way,ligation
R1421	T5001	T5000	punct	-,way
R1422	T5002	T4998	prep	between,ligation
R1423	T5003	T5004	det	a,pBS
R1424	T5004	T5002	pobj	pBS,between
R1425	T5005	T5004	amod	modified,pBS
R1426	T5006	T5004	acl	digested,pBS
R1427	T5007	T5006	agent	by,digested
R1428	T5008	T5007	pobj	HindIII,by
R1429	T5009	T5008	cc	and,HindIII
R143	T594	T590	prep	of,out
R1430	T5010	T5008	conj	NotI,HindIII
R1431	T5011	T5004	punct	", ",pBS
R1432	T5012	T5013	det	a,fragment
R1433	T5013	T5004	conj	fragment,pBS
R1434	T5014	T5015	compound	HindIII,EcoRI
R1435	T5015	T5013	compound	EcoRI,fragment
R1436	T5016	T5015	punct	-,EcoRI
R1437	T5017	T5013	prep	from,fragment
R1438	T5018	T5017	pobj	Trp53,from
R1439	T5019	T5013	prep	for,fragment
R144	T595	T596	det	a,mutation
R1440	T5020	T5021	nummod	3,homology
R1441	T5021	T5019	pobj	homology,for
R1442	T5022	T5020	punct	′,3
R1443	T5023	T5013	cc	and,fragment
R1444	T5024	T5025	det	an,fragment
R1445	T5025	T5013	conj	fragment,fragment
R1446	T5026	T5027	compound	EcoRI,NotI
R1447	T5027	T5025	compound	NotI,fragment
R1448	T5028	T5027	punct	-,NotI
R1449	T5029	T5025	acl	containing,fragment
R145	T596	T594	pobj	mutation,of
R1450	T5030	T5031	det	the,gene
R1451	T5031	T5029	dobj	gene,containing
R1452	T5032	T5031	compound	DTA,gene
R1453	T5033	T5025	punct	", ",fragment
R1454	T5034	T5025	prep	from,fragment
R1455	T5035	T5036	compound	plasmid,pgkdtabpa
R1456	T5036	T5034	pobj	pgkdtabpa,from
R1457	T5037	T5025	punct	(,fragment
R1458	T5038	T5039	amod	kind,gift
R1459	T5039	T5025	appos	gift,fragment
R146	T597	T596	amod	recessive,mutation
R1460	T5040	T5039	prep	of,gift
R1461	T5041	T5042	compound	P.,Soriano
R1462	T5042	T5040	pobj	Soriano,of
R1463	T5043	T4969	punct	),performed
R1464	T5044	T4969	punct	", ",performed
R1465	T5045	T4969	advcl	leading,performed
R1466	T5046	T5045	prep	to,leading
R1467	T5047	T5048	nmod	plasmid,DTA
R1468	T5048	T5046	pobj	DTA,to
R1469	T5049	T5048	nummod	3,DTA
R147	T598	T590	prep	after,out
R1470	T5050	T5049	punct	′,3
R1471	T5051	T5049	punct	+,3
R1472	T5052	T4969	cc	and,performed
R1473	T5053	T5054	punct	(,ii
R1474	T5054	T5055	meta	ii,deleted
R1475	T5055	T4969	conj	deleted,performed
R1476	T5056	T5054	punct	),ii
R1477	T5057	T5058	mark	because,contains
R1478	T5058	T5055	advcl	contains,deleted
R1479	T5059	T5060	det	the,region
R148	T599	T600	det	a,exchange
R1480	T5060	T5058	nsubj	region,contains
R1481	T5061	T5062	compound	Bsu36I,EcoRI
R1482	T5062	T5060	compound	EcoRI,region
R1483	T5063	T5062	punct	-,EcoRI
R1484	T5064	T5060	advmod	downstream,region
R1485	T5065	T5064	prep	of,downstream
R1486	T5066	T5065	pobj	p53,of
R1487	T5067	T5068	amod	repetitive,sequences
R1488	T5068	T5058	dobj	sequences,contains
R1489	T5069	T5070	punct	(,F.
R149	T600	T598	pobj	exchange,after
R1490	T5070	T5058	meta	F.,contains
R1491	T5071	T5070	nmod	Toledo,F.
R1492	T5072	T5070	cc	and,F.
R1493	T5073	T5070	conj	G.,F.
R1494	T5074	T5073	nmod	M.,G.
R1495	T5075	T5073	nmod	Wahl,G.
R1496	T5076	T5073	punct	", ",G.
R1497	T5077	T5078	amod	unpublished,data
R1498	T5078	T5073	conj	data,G.
R1499	T5079	T5078	punct	),data
R15	T453	T450	conj	somatic,ES
R150	T601	T600	amod	single,exchange
R1500	T5080	T5055	punct	", ",deleted
R1501	T5081	T5055	nsubj	we,deleted
R1502	T5082	T5055	advmod	later,deleted
R1503	T5083	T5084	det	this,region
R1504	T5084	T5055	dobj	region,deleted
R1505	T5085	T5055	punct	", ",deleted
R1506	T5086	T5087	aux	to,obtain
R1507	T5087	T5055	advcl	obtain,deleted
R1508	T5088	T5089	nmod	plasmid,DTA
R1509	T5089	T5087	dobj	DTA,obtain
R151	T602	T568	punct	.,provide
R1510	T5090	T5089	nummod	3,DTA
R1511	T5091	T5090	punct	′,3
R1512	T5092	T5090	punct	+,3
R1513	T5093	T4969	punct	.,performed
R1514	T5095	T5096	det	The,homology
R1515	T5096	T5099	nsubj	homology,consists
R1516	T5097	T5096	nummod	5,homology
R1517	T5098	T5097	punct	′,5
R1518	T5100	T5099	prep	of,consists
R1519	T5101	T5102	det	a,fragment
R152	T604	T605	compound	RMCE,ASAP
R1520	T5102	T5100	pobj	fragment,of
R1521	T5103	T5104	nummod	3.4,kb
R1522	T5104	T5105	npadvmod	kb,long
R1523	T5105	T5102	amod	long,fragment
R1524	T5106	T5105	punct	-,long
R1525	T5107	T5108	compound	BamHI,PmlI
R1526	T5108	T5102	compound	PmlI,fragment
R1527	T5109	T5108	punct	-,PmlI
R1528	T5110	T5102	prep	from,fragment
R1529	T5111	T5110	pobj	intron,from
R153	T605	T607	nsubj	ASAP,combines
R1530	T5112	T5111	nummod	1,intron
R1531	T5113	T5111	prep	of,intron
R1532	T5114	T5113	pobj	p53,of
R1533	T5115	T5102	acl	cloned,fragment
R1534	T5116	T5115	prep	in,cloned
R1535	T5117	T5118	det	a,pBS
R1536	T5118	T5116	pobj	pBS,in
R1537	T5119	T5118	amod	modified,pBS
R1538	T5120	T5118	punct	(,pBS
R1539	T5121	T5122	compound	plasmid,p5
R154	T606	T605	punct	-,ASAP
R1540	T5122	T5118	appos	p5,pBS
R1541	T5123	T5122	punct	′,p5
R1542	T5124	T5099	punct	),consists
R1543	T5125	T5099	punct	.,consists
R1544	T5127	T5128	advmod	Finally,assembled
R1545	T5129	T5128	punct	", ",assembled
R1546	T5130	T5131	amod	appropriate,fragments
R1547	T5131	T5128	nsubjpass	fragments,assembled
R1548	T5132	T5131	prep	from,fragments
R1549	T5133	T5134	compound	plasmids,p5
R155	T608	T609	amod	inverted,sites
R1550	T5134	T5132	pobj	p5,from
R1551	T5135	T5134	punct	′,p5
R1552	T5136	T5134	punct	", ",p5
R1553	T5137	T5138	compound	L3,1L
R1554	T5138	T5134	conj	1L,p5
R1555	T5139	T5138	punct	-,1L
R1556	T5140	T5138	compound	p53PmlEagPuroΔTK,1L
R1557	T5141	T5138	punct	-,1L
R1558	T5142	T5138	punct	", ",1L
R1559	T5143	T5138	cc	and,1L
R156	T609	T607	dobj	sites,combines
R1560	T5144	T5145	nummod	3,DTA
R1561	T5145	T5138	conj	DTA,1L
R1562	T5146	T5144	punct	′,3
R1563	T5147	T5144	punct	+,3
R1564	T5148	T5128	auxpass	were,assembled
R1565	T5149	T5128	prep	in,assembled
R1566	T5150	T5151	det	a,plasmid
R1567	T5151	T5149	pobj	plasmid,in
R1568	T5152	T5151	amod	modified,plasmid
R1569	T5153	T5151	compound	pSP72,plasmid
R157	T610	T609	amod	heterologous,sites
R1570	T5154	T5155	punct	(,Promega
R1571	T5155	T5151	parataxis	Promega,plasmid
R1572	T5156	T5155	punct	),Promega
R1573	T5157	T5128	punct	.,assembled
R1574	T5159	T5160	compound	Plasmid,Flox
R1575	T5160	T5161	nsubjpass	Flox,verified
R1576	T5162	T5160	punct	", ",Flox
R1577	T5163	T5164	det	the,construct
R1578	T5164	T5160	appos	construct,Flox
R1579	T5165	T5164	amod	resulting,construct
R158	T611	T609	compound	recombinase,sites
R1580	T5166	T5164	compound	targeting,construct
R1581	T5167	T5161	punct	", ",verified
R1582	T5168	T5161	auxpass	was,verified
R1583	T5169	T5161	prep	by,verified
R1584	T5170	T5171	compound	restriction,analysis
R1585	T5171	T5169	pobj	analysis,by
R1586	T5172	T5161	punct	", ",verified
R1587	T5173	T5161	advmod	then,verified
R1588	T5174	T5161	dep	sequenced,verified
R1589	T5175	T5174	advcl	using,sequenced
R159	T612	T609	compound	target,sites
R1590	T5176	T5177	nummod	30,primers
R1591	T5177	T5175	dobj	primers,using
R1592	T5178	T5177	acl	chosen,primers
R1593	T5179	T5180	aux	to,verify
R1594	T5180	T5178	advcl	verify,chosen
R1595	T5181	T5180	advmod	precisely,verify
R1596	T5182	T5183	det	all,sequences
R1597	T5183	T5180	dobj	sequences,verify
R1598	T5184	T5185	compound	p53,coding
R1599	T5185	T5183	compound	coding,sequences
R16	T454	T455	aux	to,accelerate
R160	T613	T609	punct	", ",sites
R1600	T5186	T5183	punct	", ",sequences
R1601	T5187	T5188	det	all,junctions
R1602	T5188	T5183	conj	junctions,sequences
R1603	T5189	T5190	compound	exon,intron
R1604	T5190	T5188	compound	intron,junctions
R1605	T5191	T5190	punct	–,intron
R1606	T5192	T5188	cc	and,junctions
R1607	T5193	T5194	det	the,sequences
R1608	T5194	T5188	conj	sequences,junctions
R1609	T5195	T5194	prep	at,sequences
R161	T614	T615	det	a,marker
R1610	T5196	T5195	cc	and,at
R1611	T5197	T5195	conj	around,at
R1612	T5198	T5199	det	the,sites
R1613	T5199	T5197	pobj	sites,around
R1614	T5200	T5199	compound	loxP,sites
R1615	T5201	T5161	punct	.,verified
R1618	T5752	T5753	compound	Exchange,constructs
R1619	T5754	T5753	punct	: ,constructs
R162	T615	T609	appos	marker,sites
R1620	T5755	T5753	acl	making,constructs
R1621	T5756	T5757	det	the,p53GFP
R1622	T5757	T5758	nmod	p53GFP,plasmids
R1623	T5758	T5755	dobj	plasmids,making
R1624	T5759	T5757	cc	and,p53GFP
R1625	T5760	T5761	compound	p53,PGFP
R1626	T5761	T5757	conj	PGFP,p53GFP
R1627	T5762	T5761	compound	Δ,PGFP
R1628	T5764	T5765	aux	To,make
R1629	T5765	T5766	advcl	make,subcloned
R163	T616	T617	amod	positive,negative
R1630	T5767	T5768	det	a,protein
R1631	T5768	T5765	dobj	protein,make
R1632	T5769	T5770	compound	p53,GFP
R1633	T5770	T5768	compound	GFP,protein
R1634	T5771	T5770	punct	-,GFP
R1635	T5772	T5768	compound	fusion,protein
R1636	T5773	T5766	punct	", ",subcloned
R1637	T5774	T5766	nsubj	we,subcloned
R1638	T5775	T5766	advmod	first,subcloned
R1639	T5776	T5777	det	a,fragment
R164	T617	T619	amod	negative,selection
R1640	T5777	T5766	dobj	fragment,subcloned
R1641	T5778	T5779	compound	SacII,HindIII
R1642	T5779	T5777	compound	HindIII,fragment
R1643	T5780	T5779	punct	-,HindIII
R1644	T5781	T5777	prep	of,fragment
R1645	T5782	T5783	det	the,locus
R1646	T5783	T5781	pobj	locus,of
R1647	T5784	T5783	compound	p53,locus
R1648	T5785	T5777	punct	(,fragment
R1649	T5786	T5777	acl	corresponding,fragment
R165	T618	T617	punct	/,negative
R1650	T5787	T5786	prep	to,corresponding
R1651	T5788	T5787	pobj	part,to
R1652	T5789	T5788	prep	of,part
R1653	T5790	T5789	pobj	exon,of
R1654	T5791	T5790	nummod	10,exon
R1655	T5792	T5786	prep	to,corresponding
R1656	T5793	T5792	pobj	sequences,to
R1657	T5794	T5793	advmod	downstream,sequences
R1658	T5795	T5794	prep	of,downstream
R1659	T5796	T5797	det	the,gene
R166	T619	T615	compound	selection,marker
R1660	T5797	T5795	pobj	gene,of
R1661	T5798	T5766	punct	),subcloned
R1662	T5799	T5766	prep	into,subcloned
R1663	T5800	T5799	pobj	pBS,into
R1664	T5801	T5766	punct	", ",subcloned
R1665	T5802	T5803	advmod	then,mutated
R1666	T5803	T5766	dep	mutated,subcloned
R1667	T5804	T5805	det	the,site
R1668	T5805	T5803	dobj	site,mutated
R1669	T5806	T5805	compound	HindIII,site
R167	T620	T621	dep	that,preserves
R1670	T5807	T5803	prep	into,mutated
R1671	T5808	T5809	det	a,site
R1672	T5809	T5807	pobj	site,into
R1673	T5810	T5809	compound	FseI,site
R1674	T5811	T5766	punct	.,subcloned
R1675	T5813	T5814	nsubj	We,mutated
R1676	T5815	T5814	advmod	next,mutated
R1677	T5816	T5817	det	the,part
R1678	T5817	T5814	dobj	part,mutated
R1679	T5818	T5819	npadvmod	C,terminal
R168	T621	T615	relcl	preserves,marker
R1680	T5819	T5817	amod	terminal,part
R1681	T5820	T5819	punct	-,terminal
R1682	T5821	T5817	prep	of,part
R1683	T5822	T5823	det	the,gene
R1684	T5823	T5821	pobj	gene,of
R1685	T5824	T5823	compound	p53,gene
R1686	T5825	T5814	prep	in,mutated
R1687	T5826	T5827	nummod	two,rounds
R1688	T5827	T5825	pobj	rounds,in
R1689	T5828	T5827	prep	of,rounds
R169	T622	T623	det	the,capacity
R1690	T5829	T5830	compound	PCR,mutagenesis
R1691	T5830	T5828	pobj	mutagenesis,of
R1692	T5831	T5814	punct	", ",mutated
R1693	T5832	T5833	advmod	first,with
R1694	T5833	T5814	prep	with,mutated
R1695	T5834	T5835	nmod	primers,GGGCCTGACTCAGACGGATCCCCTCTGCATCCCGTC
R1696	T5835	T5833	pobj	GGGCCTGACTCAGACGGATCCCCTCTGCATCCCGTC,with
R1697	T5836	T5835	nummod	5,GGGCCTGACTCAGACGGATCCCCTCTGCATCCCGTC
R1698	T5837	T5836	punct	′,5
R1699	T5838	T5835	punct	-,GGGCCTGACTCAGACGGATCCCCTCTGCATCCCGTC
R17	T455	T446	advcl	accelerate,method
R170	T623	T621	dobj	capacity,preserves
R1700	T5839	T5835	punct	-,GGGCCTGACTCAGACGGATCCCCTCTGCATCCCGTC
R1701	T5840	T5835	nummod	3,GGGCCTGACTCAGACGGATCCCCTCTGCATCCCGTC
R1702	T5841	T5835	punct	′,GGGCCTGACTCAGACGGATCCCCTCTGCATCCCGTC
R1703	T5842	T5835	cc	and,GGGCCTGACTCAGACGGATCCCCTCTGCATCCCGTC
R1704	T5843	T5844	nummod	5,GACGGGATGCAGAGGGGATCCGTCTGAGTCAGGCCC
R1705	T5844	T5835	conj	GACGGGATGCAGAGGGGATCCGTCTGAGTCAGGCCC,GGGCCTGACTCAGACGGATCCCCTCTGCATCCCGTC
R1706	T5845	T5843	punct	′,5
R1707	T5846	T5844	punct	-,GACGGGATGCAGAGGGGATCCGTCTGAGTCAGGCCC
R1708	T5847	T5844	punct	-,GACGGGATGCAGAGGGGATCCGTCTGAGTCAGGCCC
R1709	T5848	T5844	nummod	3,GACGGGATGCAGAGGGGATCCGTCTGAGTCAGGCCC
R171	T624	T623	compound	germline,capacity
R1710	T5849	T5844	punct	′,GACGGGATGCAGAGGGGATCCGTCTGAGTCAGGCCC
R1711	T5850	T5835	punct	", ",GGGCCTGACTCAGACGGATCCCCTCTGCATCCCGTC
R1712	T5851	T5852	dep	which,removed
R1713	T5852	T5835	relcl	removed,GGGCCTGACTCAGACGGATCCCCTCTGCATCCCGTC
R1714	T5853	T5854	det	the,codon
R1715	T5854	T5852	dobj	codon,removed
R1716	T5855	T5854	compound	stop,codon
R1717	T5856	T5852	cc	and,removed
R1718	T5857	T5852	conj	introduced,removed
R1719	T5858	T5859	det	a,site
R172	T625	T623	prep	of,capacity
R1720	T5859	T5857	dobj	site,introduced
R1721	T5860	T5859	compound	BamHI,site
R1722	T5861	T5833	punct	", ",with
R1723	T5862	T5863	advmod	then,with
R1724	T5863	T5833	prep	with,with
R1725	T5864	T5865	nmod	primers,GACGGATCCCCTCTGAATTCCGTCCCCATCACCA
R1726	T5865	T5863	pobj	GACGGATCCCCTCTGAATTCCGTCCCCATCACCA,with
R1727	T5866	T5865	nummod	5,GACGGATCCCCTCTGAATTCCGTCCCCATCACCA
R1728	T5867	T5866	punct	′,5
R1729	T5868	T5865	punct	-,GACGGATCCCCTCTGAATTCCGTCCCCATCACCA
R173	T626	T627	compound	ES,cells
R1730	T5869	T5865	punct	-,GACGGATCCCCTCTGAATTCCGTCCCCATCACCA
R1731	T5870	T5865	nummod	3,GACGGATCCCCTCTGAATTCCGTCCCCATCACCA
R1732	T5871	T5865	punct	′,GACGGATCCCCTCTGAATTCCGTCCCCATCACCA
R1733	T5872	T5865	cc	and,GACGGATCCCCTCTGAATTCCGTCCCCATCACCA
R1734	T5873	T5874	nummod	5,TGGTGATGGGGACGGAATACAGAGGGGATCCGTC
R1735	T5874	T5865	conj	TGGTGATGGGGACGGAATACAGAGGGGATCCGTC,GACGGATCCCCTCTGAATTCCGTCCCCATCACCA
R1736	T5875	T5873	punct	′,5
R1737	T5876	T5874	punct	-,TGGTGATGGGGACGGAATACAGAGGGGATCCGTC
R1738	T5877	T5874	punct	-,TGGTGATGGGGACGGAATACAGAGGGGATCCGTC
R1739	T5878	T5874	nummod	3,TGGTGATGGGGACGGAATACAGAGGGGATCCGTC
R174	T627	T625	pobj	cells,of
R1740	T5879	T5874	punct	′,TGGTGATGGGGACGGAATACAGAGGGGATCCGTC
R1741	T5880	T5865	punct	", ",GACGGATCCCCTCTGAATTCCGTCCCCATCACCA
R1742	T5881	T5882	dep	which,introduced
R1743	T5882	T5865	relcl	introduced,GACGGATCCCCTCTGAATTCCGTCCCCATCACCA
R1744	T5883	T5884	det	an,site
R1745	T5884	T5882	dobj	site,introduced
R1746	T5885	T5884	compound	EcoRI,site
R1747	T5886	T5814	punct	.,mutated
R1748	T5888	T5889	nsubj	We,verified
R1749	T5890	T5891	det	the,sequence
R175	T628	T623	punct	", ",capacity
R1750	T5891	T5889	dobj	sequence,verified
R1751	T5892	T5891	prep	from,sequence
R1752	T5893	T5894	det	the,plasmid
R1753	T5894	T5892	pobj	plasmid,from
R1754	T5895	T5894	amod	mutated,plasmid
R1755	T5896	T5889	punct	", ",verified
R1756	T5897	T5898	advmod	then,digested
R1757	T5898	T5889	dep	digested,verified
R1758	T5899	T5898	dobj	it,digested
R1759	T5900	T5898	prep	with,digested
R176	T629	T623	cc	and,capacity
R1760	T5901	T5900	pobj	BamHI,with
R1761	T5902	T5901	cc	and,BamHI
R1762	T5903	T5901	conj	EcoRI,BamHI
R1763	T5904	T5889	punct	", ",verified
R1764	T5905	T5906	aux	to,insert
R1765	T5906	T5889	advcl	insert,verified
R1766	T5907	T5906	prep	in,insert
R1767	T5908	T5909	compound	frame,GFP
R1768	T5909	T5910	compound	GFP,sequences
R1769	T5910	T5907	pobj	sequences,in
R177	T630	T631	det	the,power
R1770	T5911	T5910	prep	from,sequences
R1771	T5912	T5913	det	a,fragment
R1772	T5913	T5911	pobj	fragment,from
R1773	T5914	T5915	compound	Bam,EcoRI
R1774	T5915	T5913	compound	EcoRI,fragment
R1775	T5916	T5915	compound	HI,EcoRI
R1776	T5917	T5915	punct	-,EcoRI
R1777	T5918	T5913	prep	of,fragment
R1778	T5919	T5920	compound	plasmid,GFP
R1779	T5920	T5918	pobj	GFP,of
R178	T631	T623	conj	power,capacity
R1780	T5921	T5920	compound	phr,GFP
R1781	T5922	T5920	punct	-,GFP
R1782	T5923	T5920	punct	-,GFP
R1783	T5924	T5920	nummod	1,GFP
R1784	T5925	T5926	punct	(,Stratagene
R1785	T5926	T5913	parataxis	Stratagene,fragment
R1786	T5927	T5926	punct	),Stratagene
R1787	T5928	T5889	punct	.,verified
R1788	T5930	T5931	nsubj	We,verified
R1789	T5932	T5933	det	the,sequence
R179	T632	T631	prep	of,power
R1790	T5933	T5931	dobj	sequence,verified
R1791	T5934	T5933	prep	of,sequence
R1792	T5935	T5936	det	this,fragment
R1793	T5936	T5934	pobj	fragment,of
R1794	T5937	T5938	compound	p53,GFP
R1795	T5938	T5940	compound	GFP,fusion
R1796	T5939	T5938	punct	-,GFP
R1797	T5940	T5936	compound	fusion,fragment
R1798	T5941	T5931	punct	", ",verified
R1799	T5942	T5943	advmod	then,swapped
R18	T456	T457	compound	phenotype,analyses
R180	T633	T634	compound	mouse,genetics
R1800	T5943	T5931	dep	swapped,verified
R1801	T5944	T5943	dobj	it,swapped
R1802	T5945	T5943	prep	in,swapped
R1803	T5946	T5947	det	the,plasmid
R1804	T5947	T5945	pobj	plasmid,in
R1805	T5948	T5949	compound	L3,1L
R1806	T5949	T5947	compound	1L,plasmid
R1807	T5950	T5949	punct	-,1L
R1808	T5951	T5949	compound	p53PmlEagPuroΔTK,1L
R1809	T5952	T5949	punct	-,1L
R181	T634	T632	pobj	genetics,of
R1810	T5953	T5954	punct	(,see
R1811	T5954	T5943	parataxis	see,swapped
R1812	T5955	T5954	advmod	above,see
R1813	T5956	T5954	punct	),see
R1814	T5957	T5943	prep	by,swapped
R1815	T5958	T5959	nmod	HindIII,digestion
R1816	T5959	T5957	pobj	digestion,by
R1817	T5960	T5958	cc	and,HindIII
R1818	T5961	T5958	conj	FseI,HindIII
R1819	T5962	T5943	punct	", ",swapped
R182	T635	T607	punct	.,combines
R1820	T5963	T5943	advcl	resulting,swapped
R1821	T5964	T5963	prep	in,resulting
R1822	T5965	T5966	det	the,construct
R1823	T5966	T5964	pobj	construct,in
R1824	T5967	T5968	compound	p53GFP,exchange
R1825	T5968	T5966	compound	exchange,construct
R1826	T5969	T5966	punct	", ",construct
R1827	T5970	T5971	det	the,sequence
R1828	T5971	T5966	appos	sequence,construct
R1829	T5972	T5973	dep	which,verified
R183	T637	T638	det	These,principles
R1830	T5973	T5971	relcl	verified,sequence
R1831	T5974	T5973	auxpass	was,verified
R1832	T5975	T5973	prep	before,verified
R1833	T5976	T5975	pobj	use,before
R1834	T5977	T5931	punct	.,verified
R1835	T5979	T5980	det	The,construct
R1836	T5980	T5983	nsubjpass	construct,engineered
R1837	T5981	T5982	compound	p53ΔPGFP,exchange
R1838	T5982	T5980	compound	exchange,construct
R1839	T5984	T5983	auxpass	was,engineered
R184	T638	T640	nsubj	principles,make
R1840	T5985	T5983	prep	by,engineered
R1841	T5986	T5985	pcomp	combining,by
R1842	T5987	T5986	dobj	sequences,combining
R1843	T5988	T5987	prep	from,sequences
R1844	T5989	T5990	det	the,plasmid
R1845	T5990	T5988	pobj	plasmid,from
R1846	T5991	T5990	compound	p53GFP,plasmid
R1847	T5992	T5990	compound	exchange,plasmid
R1848	T5993	T5987	cc	and,sequences
R1849	T5994	T5987	conj	sequences,sequences
R185	T639	T638	amod	general,principles
R1850	T5995	T5994	prep	from,sequences
R1851	T5996	T5997	det	the,construct
R1852	T5997	T5995	pobj	construct,from
R1853	T5998	T5997	compound	p53ΔP,construct
R1854	T5999	T5997	compound	targeting,construct
R1855	T6000	T5987	acl	described,sequences
R1856	T6001	T6000	advmod	recently,described
R1857	T6002	T6003	punct	(,5
R1858	T6003	T5986	parataxis	5,combining
R1859	T6004	T6003	punct	),5
R186	T641	T640	aux	should,make
R1860	T6005	T5983	punct	.,engineered
R1861	T6007	T6008	poss	Its,sequence
R1862	T6008	T6009	nsubjpass	sequence,verified
R1863	T6010	T6009	auxpass	was,verified
R1864	T6011	T6009	advmod	also,verified
R1865	T6012	T6009	prep	before,verified
R1866	T6013	T6012	pobj	use,before
R1867	T6014	T6009	punct	.,verified
R1868	T6353	T6352	cc	and,Sequences
R1869	T6354	T6352	conj	use,Sequences
R187	T642	T643	compound	RMCE,ASAP
R1870	T6355	T6352	prep	of,Sequences
R1871	T6356	T6357	compound	PCR,primers
R1872	T6357	T6355	pobj	primers,of
R1873	T6359	T6360	meta	a,CCCCGGCCCTCACCCTCATCTTCG
R1874	T6360	T6365	nsubj	CCCCGGCCCTCACCCTCATCTTCG,amplify
R1875	T6361	T6359	punct	: ,a
R1876	T6362	T6360	nummod	5,CCCCGGCCCTCACCCTCATCTTCG
R1877	T6363	T6362	punct	′,5
R1878	T6364	T6360	punct	-,CCCCGGCCCTCACCCTCATCTTCG
R1879	T6365	T6489	ccomp	amplify,amplify
R188	T643	T645	nsubj	ASAP,applicable
R1880	T6366	T6360	punct	-,CCCCGGCCCTCACCCTCATCTTCG
R1881	T6367	T6360	nummod	3,CCCCGGCCCTCACCCTCATCTTCG
R1882	T6368	T6360	punct	′,CCCCGGCCCTCACCCTCATCTTCG
R1883	T6369	T6360	punct	", ",CCCCGGCCCTCACCCTCATCTTCG
R1884	T6370	T6360	prep	from,CCCCGGCCCTCACCCTCATCTTCG
R1885	T6371	T6372	det	the,gene
R1886	T6372	T6370	pobj	gene,from
R1887	T6373	T6372	compound	PuΔTK,gene
R1888	T6374	T6360	punct	", ",CCCCGGCCCTCACCCTCATCTTCG
R1889	T6375	T6360	appos	assays,CCCCGGCCCTCACCCTCATCTTCG
R189	T644	T643	punct	-,ASAP
R1890	T6376	T6375	acl	targeting,assays
R1891	T6377	T6376	prep	of,targeting
R1892	T6378	T6379	compound	Flox,plasmid
R1893	T6379	T6377	pobj	plasmid,of
R1894	T6380	T6360	punct	;,CCCCGGCCCTCACCCTCATCTTCG
R1895	T6381	T6382	meta	b,AACAAAACAAAACAGCAGCAACAA
R1896	T6382	T6360	conj	AACAAAACAAAACAGCAGCAACAA,CCCCGGCCCTCACCCTCATCTTCG
R1897	T6383	T6381	punct	: ,b
R1898	T6384	T6382	nummod	5,AACAAAACAAAACAGCAGCAACAA
R1899	T6385	T6384	punct	′,5
R19	T457	T455	dobj	analyses,accelerate
R190	T645	T640	ccomp	applicable,make
R1900	T6386	T6382	punct	-,AACAAAACAAAACAGCAGCAACAA
R1901	T6387	T6382	punct	-,AACAAAACAAAACAGCAGCAACAA
R1902	T6388	T6382	nummod	3,AACAAAACAAAACAGCAGCAACAA
R1903	T6389	T6382	punct	′,AACAAAACAAAACAGCAGCAACAA
R1904	T6390	T6382	punct	", ",AACAAAACAAAACAGCAGCAACAA
R1905	T6391	T6382	prep	from,AACAAAACAAAACAGCAGCAACAA
R1906	T6392	T6391	pobj	sequences,from
R1907	T6393	T6394	advmod	downstream,outside
R1908	T6394	T6392	prep	outside,sequences
R1909	T6395	T6393	prep	of,downstream
R191	T646	T645	prep	to,applicable
R1910	T6396	T6397	det	the,gene
R1911	T6397	T6395	pobj	gene,of
R1912	T6398	T6397	compound	p53,gene
R1913	T6399	T6394	cc	and,outside
R1914	T6400	T6401	compound	Flox,sequences
R1915	T6401	T6394	pobj	sequences,outside
R1916	T6402	T6382	punct	", ",AACAAAACAAAACAGCAGCAACAA
R1917	T6403	T6382	appos	assays,AACAAAACAAAACAGCAGCAACAA
R1918	T6404	T6403	acl	targeting,assays
R1919	T6405	T6404	prep	of,targeting
R192	T647	T648	det	any,locus
R1920	T6406	T6405	pobj	Flox,of
R1921	T6407	T6406	cc	and,Flox
R1922	T6408	T6406	conj	RMCE,Flox
R1923	T6409	T6403	prep	with,assays
R1924	T6410	T6411	nmod	p53GFP,plasmids
R1925	T6411	T6409	pobj	plasmids,with
R1926	T6412	T6410	cc	or,p53GFP
R1927	T6413	T6410	conj	p53ΔPGFP,p53GFP
R1928	T6414	T6382	punct	;,AACAAAACAAAACAGCAGCAACAA
R1929	T6415	T6416	meta	c,TGAAGAGCAAGGGCGTGGTGAAGGA
R193	T648	T646	pobj	locus,to
R1930	T6416	T6382	conj	TGAAGAGCAAGGGCGTGGTGAAGGA,AACAAAACAAAACAGCAGCAACAA
R1931	T6417	T6415	punct	: ,c
R1932	T6418	T6416	nummod	5,TGAAGAGCAAGGGCGTGGTGAAGGA
R1933	T6419	T6418	punct	′,5
R1934	T6420	T6416	punct	-,TGAAGAGCAAGGGCGTGGTGAAGGA
R1935	T6421	T6416	punct	-,TGAAGAGCAAGGGCGTGGTGAAGGA
R1936	T6422	T6416	nummod	3,TGAAGAGCAAGGGCGTGGTGAAGGA
R1937	T6423	T6416	punct	′,TGAAGAGCAAGGGCGTGGTGAAGGA
R1938	T6424	T6416	punct	", ",TGAAGAGCAAGGGCGTGGTGAAGGA
R1939	T6425	T6416	prep	from,TGAAGAGCAAGGGCGTGGTGAAGGA
R194	T649	T640	punct	.,make
R1940	T6426	T6427	compound	GFP,sequences
R1941	T6427	T6425	pobj	sequences,from
R1942	T6428	T6416	punct	", ",TGAAGAGCAAGGGCGTGGTGAAGGA
R1943	T6429	T6430	compound	assays,RMCE
R1944	T6430	T6416	appos	RMCE,TGAAGAGCAAGGGCGTGGTGAAGGA
R1945	T6431	T6430	prep	with,RMCE
R1946	T6432	T6433	compound	p53GFP,orp53ΔPGFP
R1947	T6433	T6434	compound	orp53ΔPGFP,plasmids
R1948	T6434	T6431	pobj	plasmids,with
R1949	T6435	T6416	punct	;,TGAAGAGCAAGGGCGTGGTGAAGGA
R195	T440	T441	compound	RMCE,ASAP
R1950	T6436	T6437	meta	d,CAAAAAATGGAAGGAAATCAGGAACTAA
R1951	T6437	T6416	conj	CAAAAAATGGAAGGAAATCAGGAACTAA,TGAAGAGCAAGGGCGTGGTGAAGGA
R1952	T6438	T6436	punct	: ,d
R1953	T6439	T6437	nummod	5,CAAAAAATGGAAGGAAATCAGGAACTAA
R1954	T6440	T6439	punct	′,5
R1955	T6441	T6437	punct	-,CAAAAAATGGAAGGAAATCAGGAACTAA
R1956	T6442	T6437	punct	-,CAAAAAATGGAAGGAAATCAGGAACTAA
R1957	T6443	T6437	nummod	3,CAAAAAATGGAAGGAAATCAGGAACTAA
R1958	T6444	T6437	punct	′,CAAAAAATGGAAGGAAATCAGGAACTAA
R1959	T6445	T6437	punct	", ",CAAAAAATGGAAGGAAATCAGGAACTAA
R1960	T6446	T6437	prep	from,CAAAAAATGGAAGGAAATCAGGAACTAA
R1961	T6447	T6448	compound	p53,intron
R1962	T6448	T6446	pobj	intron,from
R1963	T6449	T6448	nummod	3,intron
R1964	T6450	T6437	punct	", ",CAAAAAATGGAAGGAAATCAGGAACTAA
R1965	T6451	T6437	cc	and,CAAAAAATGGAAGGAAATCAGGAACTAA
R1966	T6452	T6453	meta	e,TCTAGACAGAGAAAAAAGAGGCATT
R1967	T6453	T6437	conj	TCTAGACAGAGAAAAAAGAGGCATT,CAAAAAATGGAAGGAAATCAGGAACTAA
R1968	T6454	T6452	punct	: ,e
R1969	T6455	T6453	nummod	5,TCTAGACAGAGAAAAAAGAGGCATT
R1970	T6456	T6455	punct	′,5
R1971	T6457	T6453	punct	-,TCTAGACAGAGAAAAAAGAGGCATT
R1972	T6458	T6453	punct	-,TCTAGACAGAGAAAAAAGAGGCATT
R1973	T6459	T6453	nummod	3,TCTAGACAGAGAAAAAAGAGGCATT
R1974	T6460	T6453	punct	′,TCTAGACAGAGAAAAAAGAGGCATT
R1975	T6461	T6453	punct	", ",TCTAGACAGAGAAAAAAGAGGCATT
R1976	T6462	T6453	prep	from,TCTAGACAGAGAAAAAAGAGGCATT
R1977	T6463	T6464	compound	p53,intron
R1978	T6464	T6462	pobj	intron,from
R1979	T6465	T6464	nummod	4,intron
R1980	T6466	T6453	punct	", ",TCTAGACAGAGAAAAAAGAGGCATT
R1981	T6467	T6468	compound	assay,RMCE
R1982	T6468	T6453	appos	RMCE,TCTAGACAGAGAAAAAAGAGGCATT
R1983	T6469	T6468	prep	with,RMCE
R1984	T6470	T6471	compound	p53ΔPGFP,plasmid
R1985	T6471	T6469	pobj	plasmid,with
R1986	T6472	T6453	punct	;,TCTAGACAGAGAAAAAAGAGGCATT
R1987	T6473	T6474	meta	f,ATGGGAGGCTGCCAGTCCTAACCC
R1988	T6474	T6453	conj	ATGGGAGGCTGCCAGTCCTAACCC,TCTAGACAGAGAAAAAAGAGGCATT
R1989	T6475	T6473	punct	: ,f
R1990	T6476	T6474	nummod	5,ATGGGAGGCTGCCAGTCCTAACCC
R1991	T6477	T6474	punct	′,ATGGGAGGCTGCCAGTCCTAACCC
R1992	T6478	T6474	punct	-,ATGGGAGGCTGCCAGTCCTAACCC
R1993	T6479	T6474	cc	and,ATGGGAGGCTGCCAGTCCTAACCC
R1994	T6480	T6481	meta	g,GTGTTTCATTAGTTCCCCACCTTGAC
R1995	T6481	T6474	conj	GTGTTTCATTAGTTCCCCACCTTGAC,ATGGGAGGCTGCCAGTCCTAACCC
R1996	T6482	T6480	punct	: ,g
R1997	T6483	T6481	nummod	5,GTGTTTCATTAGTTCCCCACCTTGAC
R1998	T6484	T6483	punct	′,5
R1999	T6485	T6481	punct	-,GTGTTTCATTAGTTCCCCACCTTGAC
R20	T460	T461	prep	In,gained
R2000	T6486	T6481	punct	-,GTGTTTCATTAGTTCCCCACCTTGAC
R2001	T6487	T6481	nummod	3,GTGTTTCATTAGTTCCCCACCTTGAC
R2002	T6488	T6481	punct	′,GTGTTTCATTAGTTCCCCACCTTGAC
R2003	T6490	T6491	det	the,allele
R2004	T6491	T6365	dobj	allele,amplify
R2005	T6492	T6493	compound	WT,p53
R2006	T6493	T6491	compound	p53,allele
R2007	T6494	T6365	prep	according,amplify
R2008	T6495	T6494	prep	to,according
R2009	T6496	T6497	poss	Taconic,procedures
R2010	T6497	T6495	pobj	procedures,to
R2011	T6498	T6496	case	's,Taconic
R2012	T6499	T6489	punct	", ",amplify
R2013	T6500	T6501	meta	h,TTTACGGAGCCCTGGCGCTCGATGT
R2014	T6501	T6489	nsubj	TTTACGGAGCCCTGGCGCTCGATGT,amplify
R2015	T6502	T6500	punct	: ,h
R2016	T6503	T6501	nummod	5,TTTACGGAGCCCTGGCGCTCGATGT
R2017	T6504	T6501	punct	′,TTTACGGAGCCCTGGCGCTCGATGT
R2018	T6505	T6501	punct	-,TTTACGGAGCCCTGGCGCTCGATGT
R2019	T6506	T6501	punct	-,TTTACGGAGCCCTGGCGCTCGATGT
R2020	T6507	T6501	nummod	3,TTTACGGAGCCCTGGCGCTCGATGT
R2021	T6508	T6501	punct	′,TTTACGGAGCCCTGGCGCTCGATGT
R2022	T6509	T6501	cc	and,TTTACGGAGCCCTGGCGCTCGATGT
R2023	T6510	T6511	meta	i,GTGGGAGGGACAAAAGTTCGAGGCC
R2024	T6511	T6501	conj	GTGGGAGGGACAAAAGTTCGAGGCC,TTTACGGAGCCCTGGCGCTCGATGT
R2025	T6512	T6510	punct	: ,i
R2026	T6513	T6511	nummod	5,GTGGGAGGGACAAAAGTTCGAGGCC
R2027	T6514	T6511	punct	′,GTGGGAGGGACAAAAGTTCGAGGCC
R2028	T6515	T6511	punct	-,GTGGGAGGGACAAAAGTTCGAGGCC
R2029	T6516	T6511	punct	-,GTGGGAGGGACAAAAGTTCGAGGCC
R2030	T6517	T6511	nummod	3,GTGGGAGGGACAAAAGTTCGAGGCC
R2031	T6518	T6511	punct	′,GTGGGAGGGACAAAAGTTCGAGGCC
R2032	T6519	T6520	det	the,marker
R2033	T6520	T6489	dobj	marker,amplify
R2034	T6521	T6520	compound	Neo,marker
R2035	T6522	T6520	prep	in,marker
R2036	T6523	T6524	det	the,allele
R2037	T6524	T6522	pobj	allele,in
R2038	T6525	T6526	compound	p53,KO
R2039	T6526	T6524	compound	KO,allele
R204	T2571	T2572	nsubj	p53,is
R2040	T6527	T6489	prep	according,amplify
R2041	T6528	T6527	prep	to,according
R2042	T6529	T6530	poss	Taconic,procedures
R2043	T6530	T6528	pobj	procedures,to
R2044	T6531	T6529	case	's,Taconic
R2045	T6532	T6489	punct	.,amplify
R2046	T6727	T6728	compound	Cell,culture
R2047	T6728	T6729	compound	culture,conditions
R2048	T6731	T6732	amod	Primary,MEFs
R2049	T6732	T6733	nsubjpass	MEFs,cultured
R205	T2573	T2572	attr	one,is
R2050	T6734	T6732	punct	", ",MEFs
R2051	T6735	T6732	acl	isolated,MEFs
R2052	T6736	T6735	prep	from,isolated
R2053	T6737	T6738	nummod	13.5,day
R2054	T6738	T6739	compound	day,embryos
R2055	T6739	T6736	pobj	embryos,from
R2056	T6740	T6733	punct	", ",cultured
R2057	T6741	T6733	auxpass	were,cultured
R2058	T6742	T6733	prep	in,cultured
R2059	T6743	T6742	pobj	DMEM,in
R206	T2574	T2573	prep	of,one
R2060	T6744	T6733	prep	with,cultured
R2061	T6745	T6746	nummod	15,%
R2062	T6746	T6747	compound	%,FBS
R2063	T6747	T6744	pobj	FBS,with
R2064	T6748	T6747	punct	", ",FBS
R2065	T6749	T6750	nummod	100,mM
R2066	T6750	T6751	compound	mM,BME
R2067	T6751	T6747	conj	BME,FBS
R2068	T6752	T6751	punct	", ",BME
R2069	T6753	T6754	nummod	2,mM
R207	T2575	T2576	det	the,proteins
R2070	T6754	T6755	compound	mM,glutamine
R2071	T6755	T6751	conj	glutamine,BME
R2072	T6756	T6755	compound	l,glutamine
R2073	T6757	T6755	punct	-,glutamine
R2074	T6758	T6755	cc	and,glutamine
R2075	T6759	T6755	conj	antibiotics,glutamine
R2076	T6760	T6733	punct	.,cultured
R2077	T6762	T6763	nummod	129,SvJae
R2078	T6763	T6765	compound	SvJae,cells
R2079	T6764	T6763	punct	/,SvJae
R208	T2576	T2574	pobj	proteins,of
R2080	T6765	T6767	nsubjpass	cells,grown
R2081	T6766	T6765	compound	ES,cells
R2082	T6768	T6767	auxpass	were,grown
R2083	T6769	T6767	prep	in,grown
R2084	T6770	T6771	det	the,medium
R2085	T6771	T6769	pobj	medium,in
R2086	T6772	T6771	amod	same,medium
R2087	T6773	T6771	acl	supplemented,medium
R2088	T6774	T6773	prep	with,supplemented
R2089	T6775	T6776	nummod	1000,U
R209	T2577	T2578	advmod	most,highly
R2090	T6776	T6777	nmod	U,ESGRO
R2091	T6777	T6774	pobj	ESGRO,with
R2092	T6778	T6779	punct	/,ml
R2093	T6779	T6776	prep	ml,U
R2094	T6780	T6781	punct	(,Chemicon
R2095	T6781	T6777	parataxis	Chemicon,ESGRO
R2096	T6782	T6781	punct	),Chemicon
R2097	T6783	T6767	punct	", ",grown
R2098	T6784	T6767	prep	on,grown
R2099	T6785	T6786	det	a,layer
R21	T462	T463	amod	recent,years
R210	T2578	T2579	advmod	highly,analyzed
R2100	T6786	T6784	pobj	layer,on
R2101	T6787	T6786	prep	of,layer
R2102	T6788	T6789	compound	mitomycin,C
R2103	T6789	T6790	npadvmod	C,treated
R2104	T6790	T6792	amod	treated,feeders
R2105	T6791	T6790	punct	-,treated
R2106	T6792	T6787	pobj	feeders,of
R2107	T6793	T6792	nmod	SNLPuro,feeders
R2108	T6794	T6793	punct	-,SNLPuro
R2109	T6795	T6793	nummod	7,SNLPuro
R211	T2579	T2576	amod	analyzed,proteins
R2110	T6796	T6793	punct	/,SNLPuro
R2111	T6797	T6793	nummod	4,SNLPuro
R2112	T6798	T6799	punct	(,gift
R2113	T6799	T6786	parataxis	gift,layer
R2114	T6800	T6799	amod	kind,gift
R2115	T6801	T6799	prep	of,gift
R2116	T6802	T6803	compound	A.,Bradley
R2117	T6803	T6801	pobj	Bradley,of
R2118	T6804	T6799	punct	),gift
R2119	T6805	T6767	punct	.,grown
R212	T2580	T2572	prep	for,is
R2120	T6807	T6808	nsubjpass	Selections,performed
R2121	T6809	T6808	auxpass	were,performed
R2122	T6810	T6808	prep	with,performed
R2123	T6811	T6812	nummod	2,μg
R2124	T6812	T6813	nmod	μg,puromycin
R2125	T6813	T6810	pobj	puromycin,with
R2126	T6814	T6815	punct	/,ml
R2127	T6815	T6812	prep	ml,μg
R2128	T6816	T6813	punct	", ",puromycin
R2129	T6817	T6818	nummod	0.2,μM
R213	T2581	T2582	det	the,years
R2130	T6818	T6819	compound	μM,FIAU
R2131	T6819	T6813	conj	FIAU,puromycin
R2132	T6820	T6819	cc	or,FIAU
R2133	T6821	T6822	nummod	2,μM
R2134	T6822	T6823	compound	μM,ganciclovir
R2135	T6823	T6819	conj	ganciclovir,FIAU
R2136	T6824	T6808	punct	.,performed
R2137	T6929	T6930	compound	Targeting,genotyping
R2138	T6931	T6930	punct	/,genotyping
R2139	T6932	T6930	prep	of,genotyping
R214	T2582	T2580	pobj	years,for
R2140	T6933	T6934	det	the,locus
R2141	T6934	T6932	pobj	locus,of
R2142	T6935	T6936	amod	RMCE,ready
R2143	T6936	T6934	amod	ready,locus
R2144	T6937	T6936	punct	-,ready
R2145	T6939	T6940	nummod	29,SvJae
R2146	T6940	T6942	compound	SvJae,cells
R2147	T6941	T6940	punct	/,SvJae
R2148	T6942	T6944	nsubjpass	cells,electroporated
R2149	T6943	T6942	compound	ES,cells
R215	T2583	T2582	amod	past,years
R2150	T6945	T6944	auxpass	were,electroporated
R2151	T6946	T6944	prep	with,electroporated
R2152	T6947	T6948	det	the,construct
R2153	T6948	T6946	pobj	construct,with
R2154	T6949	T6948	compound	Flox,construct
R2155	T6950	T6948	acl	linearized,construct
R2156	T6951	T6950	prep	with,linearized
R2157	T6952	T6951	pobj	PmeI,with
R2158	T6953	T6944	punct	", ",electroporated
R2159	T6954	T6944	cc	and,electroporated
R216	T2584	T2582	nummod	25,years
R2160	T6955	T6956	npadvmod	puromycin,resistant
R2161	T6956	T6957	amod	resistant,clones
R2162	T6957	T6958	nsubjpass	clones,analyzed
R2163	T6958	T6944	conj	analyzed,electroporated
R2164	T6959	T6958	auxpass	were,analyzed
R2165	T6960	T6961	mark	as,described
R2166	T6961	T6958	advcl	described,analyzed
R2167	T6962	T6963	punct	(,Figure
R2168	T6963	T6958	parataxis	Figure,analyzed
R2169	T6964	T6963	nummod	2,Figure
R217	T2585	T2572	punct	.,is
R2170	T6965	T6963	punct	),Figure
R2171	T6966	T6958	punct	.,analyzed
R2172	T6968	T6969	nummod	Two,clones
R2173	T6969	T6970	nsubjpass	clones,injected
R2174	T6971	T6970	auxpass	were,injected
R2175	T6972	T6970	prep	into,injected
R2176	T6973	T6972	pobj	blastocysts,into
R2177	T6974	T6970	cc	and,injected
R2178	T6975	T6970	conj	transmitted,injected
R2179	T6976	T6975	prep	through,transmitted
R218	T2587	T2588	nsubj	Studies,established
R2180	T6977	T6978	det	the,germline
R2181	T6978	T6976	pobj	germline,through
R2182	T6979	T6970	punct	.,injected
R2183	T7193	T7192	dobj	RMCE,Performing
R2184	T7194	T7192	prep	in,Performing
R2185	T7195	T7196	compound	ES,cells
R2186	T7196	T7194	pobj	cells,in
R2187	T7198	T7199	det	A,total
R2188	T7199	T7200	nsubjpass	total,grown
R2189	T7201	T7199	prep	of,total
R219	T2589	T2587	prep	in,Studies
R2190	T7202	T7203	compound	8,105
R2191	T7203	T7205	nummod	105,cells
R2192	T7204	T7203	punct	×,105
R2193	T7205	T7201	pobj	cells,of
R2194	T7206	T7205	nmod	p53RMCE,cells
R2195	T7207	T7206	punct	/,p53RMCE
R2196	T7208	T7206	punct	+,p53RMCE
R2197	T7209	T7205	compound	ES,cells
R2198	T7210	T7200	auxpass	were,grown
R2199	T7211	T7200	prep	without,grown
R22	T463	T460	pobj	years,In
R220	T2590	T2591	amod	cultured,cells
R2200	T7212	T7211	pobj	puromycin,without
R2201	T7213	T7200	prep	for,grown
R2202	T7214	T7215	nummod	12,h
R2203	T7215	T7213	pobj	h,for
R2204	T7216	T7200	punct	", ",grown
R2205	T7217	T7200	conj	electroporated,grown
R2206	T7218	T7217	prep	with,electroporated
R2207	T7219	T7220	nummod	15,plasmid
R2208	T7220	T7218	pobj	plasmid,with
R2209	T7221	T7220	compound	μg,plasmid
R221	T2591	T2589	pobj	cells,in
R2210	T7222	T7223	compound	CMV,Cre
R2211	T7223	T7220	compound	Cre,plasmid
R2212	T7224	T7223	punct	-,Cre
R2213	T7225	T7226	punct	(,pOG231
R2214	T7226	T7220	parataxis	pOG231,plasmid
R2215	T7227	T7226	punct	),pOG231
R2216	T7228	T7220	cc	and,plasmid
R2217	T7229	T7230	nummod	200,μg
R2218	T7230	T7220	conj	μg,plasmid
R2219	T7231	T7230	prep	of,μg
R222	T2592	T2587	punct	", ",Studies
R2220	T7232	T7233	det	the,construct
R2221	T7233	T7231	pobj	construct,of
R2222	T7234	T7233	compound	exchange,construct
R2223	T7235	T7217	punct	", ",electroporated
R2224	T7236	T7217	cc	and,electroporated
R2225	T7237	T7217	conj	plated,electroporated
R2226	T7238	T7237	prep	in,plated
R2227	T7239	T7240	compound	T25,flasks
R2228	T7240	T7238	pobj	flasks,in
R2229	T7241	T7237	prep	at,plated
R223	T2593	T2594	advmod	often,relying
R2230	T7242	T7243	nummod	105,cells
R2231	T7243	T7241	pobj	cells,at
R2232	T7244	T7243	prep	per,cells
R2233	T7245	T7244	pobj	flask,per
R2234	T7246	T7200	punct	.,grown
R2235	T7248	T7249	nsubjpass	FIAU,added
R2236	T7250	T7249	auxpass	was,added
R2237	T7251	T7249	prep	to,added
R2238	T7252	T7253	det	the,medium
R2239	T7253	T7251	pobj	medium,to
R224	T2594	T2587	acl	relying,Studies
R2240	T7254	T7255	compound	3,4
R2241	T7255	T7257	nummod	4,days
R2242	T7256	T7255	punct	–,4
R2243	T7257	T7258	npadvmod	days,after
R2244	T7258	T7249	prep	after,added
R2245	T7259	T7258	pobj	electroporation,after
R2246	T7260	T7249	punct	.,added
R2247	T7262	T7263	amod	Individual,clones
R2248	T7263	T7264	nsubjpass	clones,grown
R2249	T7265	T7263	punct	", ",clones
R225	T2595	T2594	prep	on,relying
R2250	T7266	T7263	acl	picked,clones
R2251	T7267	T7268	nummod	10,days
R2252	T7268	T7266	npadvmod	days,picked
R2253	T7269	T7268	prep	after,days
R2254	T7270	T7269	pobj	electroporation,after
R2255	T7271	T7264	punct	", ",grown
R2256	T7272	T7264	auxpass	were,grown
R2257	T7273	T7264	prep	in,grown
R2258	T7274	T7275	nummod	96,well
R2259	T7275	T7277	compound	well,plates
R226	T2596	T2597	det	the,transfection
R2260	T7276	T7275	punct	-,well
R2261	T7277	T7273	pobj	plates,in
R2262	T7278	T7264	cc	and,grown
R2263	T7279	T7264	conj	expanded,grown
R2264	T7280	T7281	aux	to,generate
R2265	T7281	T7279	advcl	generate,expanded
R2266	T7282	T7283	amod	duplicate,plates
R2267	T7283	T7281	dobj	plates,generate
R2268	T7284	T7283	prep	for,plates
R2269	T7285	T7284	pobj	freezing,for
R227	T2597	T2595	pobj	transfection,on
R2270	T7286	T7285	cc	and,freezing
R2271	T7287	T7288	compound	DNA,analysis
R2272	T7288	T7285	conj	analysis,freezing
R2273	T7289	T7281	prep	by,generate
R2274	T7290	T7289	pobj	PCR,by
R2275	T7291	T7290	cc	and,PCR
R2276	T7292	T7290	conj	Southern,PCR
R2277	T7293	T7264	punct	.,grown
R2278	T7498	T7497	dobj	RMCE,Performing
R2279	T7499	T7497	prep	in,Performing
R228	T2598	T2597	prep	of,transfection
R2280	T7500	T7499	pobj	MEFs,in
R2281	T7502	T7503	det	A,total
R2282	T7503	T7504	nsubjpass	total,grown
R2283	T7505	T7503	prep	of,total
R2284	T7506	T7507	nummod	106,cells
R2285	T7507	T7505	pobj	cells,of
R2286	T7508	T7509	nmod	p53RMCE,MEFs
R2287	T7509	T7507	compound	MEFs,cells
R2288	T7510	T7509	punct	/,MEFs
R2289	T7511	T7509	punct	−,MEFs
R229	T2599	T2598	pobj	plasmids,of
R2290	T7512	T7504	auxpass	were,grown
R2291	T7513	T7504	prep	without,grown
R2292	T7514	T7513	pobj	puromycin,without
R2293	T7515	T7504	prep	for,grown
R2294	T7516	T7517	nummod	12,h
R2295	T7517	T7515	pobj	h,for
R2296	T7518	T7504	punct	", ",grown
R2297	T7519	T7504	conj	electroporated,grown
R2298	T7520	T7519	prep	with,electroporated
R2299	T7521	T7522	nummod	3,μg
R23	T464	T461	punct	", ",gained
R230	T2600	T2599	acl	expressing,plasmids
R2300	T7522	T7523	compound	μg,pOG231
R2301	T7523	T7520	pobj	pOG231,with
R2302	T7524	T7523	cc	and,pOG231
R2303	T7525	T7526	nummod	30,μg
R2304	T7526	T7527	compound	μg,construct
R2305	T7527	T7523	conj	construct,pOG231
R2306	T7528	T7527	compound	exchange,construct
R2307	T7529	T7519	punct	", ",electroporated
R2308	T7530	T7519	cc	and,electroporated
R2309	T7531	T7519	conj	plated,electroporated
R231	T2601	T2602	amod	various,mutants
R2310	T7532	T7531	prep	in,plated
R2311	T7533	T7534	det	a,dish
R2312	T7534	T7532	pobj	dish,in
R2313	T7535	T7534	amod	single,dish
R2314	T7536	T7537	nummod	10,cm
R2315	T7537	T7534	compound	cm,dish
R2316	T7538	T7534	punct	-,dish
R2317	T7539	T7531	punct	", ",plated
R2318	T7540	T7531	conj	grown,plated
R2319	T7541	T7540	prep	for,grown
R232	T2602	T2600	dobj	mutants,expressing
R2320	T7542	T7543	nummod	3,days
R2321	T7543	T7541	pobj	days,for
R2322	T7544	T7545	advmod	then,split
R2323	T7545	T7540	dep	split,grown
R2324	T7546	T7545	prep	in,split
R2325	T7547	T7548	amod	several,dishes
R2326	T7548	T7546	pobj	dishes,in
R2327	T7549	T7545	prep	at,split
R2328	T7550	T7551	nummod	105,cells
R2329	T7551	T7549	pobj	cells,at
R233	T2603	T2602	compound	p53,mutants
R2330	T7552	T7551	prep	per,cells
R2331	T7553	T7552	pobj	dish,per
R2332	T7554	T7504	punct	.,grown
R2333	T7556	T7557	nsubjpass	FIAU,added
R2334	T7558	T7556	cc	or,FIAU
R2335	T7559	T7556	conj	ganciclovir,FIAU
R2336	T7560	T7557	auxpass	was,added
R2337	T7561	T7557	prep	to,added
R2338	T7562	T7563	det	the,medium
R2339	T7563	T7561	pobj	medium,to
R234	T2604	T2588	punct	", ",established
R2340	T7564	T7565	nummod	4,days
R2341	T7565	T7566	npadvmod	days,after
R2342	T7566	T7557	prep	after,added
R2343	T7567	T7566	pobj	electroporation,after
R2344	T7568	T7557	punct	", ",added
R2345	T7569	T7557	prep	for,added
R2346	T7570	T7571	quantmod	3,4
R2347	T7571	T7573	nummod	4,days
R2348	T7572	T7571	punct	–,4
R2349	T7573	T7569	pobj	days,for
R235	T2605	T2588	aux	have,established
R2350	T7574	T7557	punct	.,added
R2351	T7576	T7577	nsubjpass	Clones,grown
R2352	T7578	T7576	punct	", ",Clones
R2353	T7579	T7576	acl	picked,Clones
R2354	T7580	T7581	nummod	10,days
R2355	T7581	T7582	npadvmod	days,after
R2356	T7582	T7579	prep	after,picked
R2357	T7583	T7582	pobj	electroporation,after
R2358	T7584	T7577	punct	", ",grown
R2359	T7585	T7577	auxpass	were,grown
R236	T2606	T2588	dobj	models,established
R2360	T7586	T7577	prep	in,grown
R2361	T7587	T7588	nummod	24,well
R2362	T7588	T7590	compound	well,plates
R2363	T7589	T7588	punct	-,well
R2364	T7590	T7586	pobj	plates,in
R2365	T7591	T7577	cc	and,grown
R2366	T7592	T7577	conj	expanded,grown
R2367	T7593	T7592	prep	for,expanded
R2368	T7594	T7593	pobj	freezing,for
R2369	T7595	T7594	cc	and,freezing
R237	T2607	T2608	aux	to,explain
R2370	T7596	T7597	compound	DNA,analysis
R2371	T7597	T7594	conj	analysis,freezing
R2372	T7598	T7577	punct	.,grown
R2373	T8070	T8071	compound	Western,blots
R2374	T8072	T8071	punct	-,blots
R2375	T8074	T8075	nsubjpass	Cells,lysed
R2376	T8076	T8074	punct	", ",Cells
R2377	T8077	T8074	amod	untreated,Cells
R2378	T8078	T8077	cc	or,untreated
R2379	T8079	T8077	conj	treated,untreated
R238	T2608	T2606	advcl	explain,models
R2380	T8080	T8079	prep	for,treated
R2381	T8081	T8082	nummod	24,h
R2382	T8082	T8080	pobj	h,for
R2383	T8083	T8079	prep	with,treated
R2384	T8084	T8085	nummod	0.5,μg
R2385	T8085	T8086	nmod	μg,adriamycin
R2386	T8086	T8083	pobj	adriamycin,with
R2387	T8087	T8088	punct	/,ml
R2388	T8088	T8085	prep	ml,μg
R2389	T8089	T8075	punct	", ",lysed
R239	T2609	T2610	advmod	how,regulated
R2390	T8090	T8075	auxpass	were,lysed
R2391	T8091	T8075	prep	on,lysed
R2392	T8092	T8093	det	the,dish
R2393	T8093	T8091	pobj	dish,on
R2394	T8094	T8075	prep	in,lysed
R2395	T8095	T8096	det	a,buffer
R2396	T8096	T8094	pobj	buffer,in
R2397	T8097	T8096	acl	consisting,buffer
R2398	T8098	T8097	prep	of,consisting
R2399	T8099	T8100	nummod	50,mM
R24	T465	T466	amod	tremendous,insight
R240	T2610	T2608	ccomp	regulated,explain
R2400	T8100	T8101	compound	mM,Tris
R2401	T8101	T8098	pobj	Tris,of
R2402	T8102	T8103	punct	(,pH
R2403	T8103	T8101	parataxis	pH,Tris
R2404	T8104	T8103	nummod	8.0,pH
R2405	T8105	T8103	punct	),pH
R2406	T8106	T8101	punct	", ",Tris
R2407	T8107	T8108	nummod	5,mM
R2408	T8108	T8109	compound	mM,EDTA
R2409	T8109	T8101	conj	EDTA,Tris
R241	T2611	T2610	nsubjpass	p53,regulated
R2410	T8110	T8109	punct	", ",EDTA
R2411	T8111	T8112	nummod	150,mM
R2412	T8112	T8113	compound	mM,NaCl
R2413	T8113	T8109	conj	NaCl,EDTA
R2414	T8114	T8113	punct	", ",NaCl
R2415	T8115	T8116	nummod	0.5,%
R2416	T8116	T8117	compound	%,40
R2417	T8117	T8113	conj	40,NaCl
R2418	T8118	T8117	compound	Nonidet,40
R2419	T8119	T8117	compound	P,40
R242	T2612	T2610	auxpass	is,regulated
R2420	T8120	T8117	punct	-,40
R2421	T8121	T8117	punct	", ",40
R2422	T8122	T8123	nummod	1,mM
R2423	T8123	T8124	compound	mM,PMSF
R2424	T8124	T8117	conj	PMSF,40
R2425	T8125	T8124	punct	", ",PMSF
R2426	T8126	T8127	nummod	1,mM
R2427	T8127	T8128	compound	mM,vanadate
R2428	T8128	T8124	conj	vanadate,PMSF
R2429	T8129	T8128	compound	sodium,vanadate
R243	T2613	T2588	punct	.,established
R2430	T8130	T8128	punct	", ",vanadate
R2431	T8131	T8132	nummod	10,mM
R2432	T8132	T8133	compound	mM,NaF
R2433	T8133	T8128	conj	NaF,vanadate
R2434	T8134	T8133	cc	and,NaF
R2435	T8135	T8136	compound	Complete,Mini
R2436	T8136	T8137	compound	Mini,Inhibitors
R2437	T8137	T8133	conj	Inhibitors,NaF
R2438	T8138	T8137	compound	Protease,Inhibitors
R2439	T8139	T8140	punct	(,Diagnostics
R244	T2615	T2616	prep	In,tested
R2440	T8140	T8137	parataxis	Diagnostics,Inhibitors
R2441	T8141	T8140	compound	Roche,Diagnostics
R2442	T8142	T8140	punct	),Diagnostics
R2443	T8143	T8075	prep	at,lysed
R2444	T8144	T8145	nummod	4,°C
R2445	T8145	T8143	pobj	°C,at
R2446	T8146	T8075	prep	for,lysed
R2447	T8147	T8148	nummod	30,min
R2448	T8148	T8146	pobj	min,for
R2449	T8149	T8075	punct	.,lysed
R245	T2617	T2618	amod	recent,years
R2450	T8151	T8152	nsubjpass	Lysates,scraped
R2451	T8153	T8152	auxpass	were,scraped
R2452	T8154	T8152	punct	", ",scraped
R2453	T8155	T8156	advmod	then,spun
R2454	T8156	T8152	dep	spun,scraped
R2455	T8157	T8156	prep	at,spun
R2456	T8158	T8159	nummod	6000,g
R2457	T8159	T8157	pobj	g,at
R2458	T8160	T8159	punct	×,g
R2459	T8161	T8156	prep	at,spun
R246	T2618	T2615	pobj	years,In
R2460	T8162	T8163	nummod	4,°C
R2461	T8163	T8161	pobj	°C,at
R2462	T8164	T8156	prep	for,spun
R2463	T8165	T8166	nummod	10,min
R2464	T8166	T8164	pobj	min,for
R2465	T8167	T8152	punct	.,scraped
R2466	T8169	T8170	compound	Protein,concentration
R2467	T8170	T8171	nsubjpass	concentration,determined
R2468	T8172	T8170	prep	in,concentration
R2469	T8173	T8174	det	the,supernatant
R247	T2619	T2616	punct	", ",tested
R2470	T8174	T8172	pobj	supernatant,in
R2471	T8175	T8171	auxpass	was,determined
R2472	T8176	T8171	advcl	using,determined
R2473	T8177	T8178	det	the,assay
R2474	T8178	T8176	dobj	assay,using
R2475	T8179	T8180	compound	Bio,Rad
R2476	T8180	T8178	compound	Rad,assay
R2477	T8181	T8180	punct	-,Rad
R2478	T8182	T8178	compound	DC,assay
R2479	T8183	T8178	compound	protein,assay
R248	T2620	T2616	nsubjpass	some,tested
R2480	T8184	T8171	punct	.,determined
R2481	T8186	T8187	nsubjpass	Lysates,separated
R2482	T8188	T8187	auxpass	were,separated
R2483	T8189	T8187	prep	on,separated
R2484	T8190	T8191	amod	single,percentage
R2485	T8191	T8192	compound	percentage,gels
R2486	T8192	T8189	pobj	gels,on
R2487	T8193	T8194	compound	SDS,PAGE
R2488	T8194	T8192	compound	PAGE,gels
R2489	T8195	T8194	punct	/,PAGE
R249	T2621	T2620	prep	of,some
R2490	T8196	T8187	punct	", ",separated
R2491	T8197	T8198	advmod	then,transferred
R2492	T8198	T8187	dep	transferred,separated
R2493	T8199	T8198	advmod	electrophoretically,transferred
R2494	T8200	T8198	prep	to,transferred
R2495	T8201	T8200	pobj	poly,to
R2496	T8202	T8203	punct	(,difluoride
R2497	T8203	T8201	parataxis	difluoride,poly
R2498	T8204	T8203	compound	vinylidene,difluoride
R2499	T8205	T8203	punct	),difluoride
R25	T466	T461	nsubjpass	insight,gained
R250	T2622	T2623	det	these,models
R2500	T8206	T8198	punct	", ",transferred
R2501	T8207	T8198	advcl	using,transferred
R2502	T8208	T8209	amod	standard,procedures
R2503	T8209	T8207	dobj	procedures,using
R2504	T8210	T8187	punct	.,separated
R2505	T8211	T8212	nsubjpass	Blots,incubated
R2506	T8214	T8212	auxpass	were,incubated
R2507	T8215	T8212	prep	in,incubated
R2508	T8216	T8217	nummod	5,%
R2509	T8217	T8218	nmod	%,milk
R251	T2623	T2621	pobj	models,of
R2510	T8218	T8215	pobj	milk,in
R2511	T8219	T8218	amod	non-fat,milk
R2512	T8220	T8218	amod	dried,milk
R2513	T8221	T8212	prep	in,incubated
R2514	T8222	T8221	pobj	TBST,in
R2515	T8223	T8224	punct	(,Tween
R2516	T8224	T8222	parataxis	Tween,TBST
R2517	T8225	T8226	nummod	0.02,M
R2518	T8226	T8227	compound	M,Tris
R2519	T8227	T8224	dep	Tris,Tween
R252	T2624	T2616	auxpass	were,tested
R2520	T8228	T8224	punct	", ",Tween
R2521	T8229	T8224	nmod	pH,Tween
R2522	T8230	T8229	nummod	7.6,pH
R2523	T8231	T8224	punct	/,Tween
R2524	T8232	T8233	nummod	0.35,M
R2525	T8233	T8234	nmod	M,NaCl
R2526	T8234	T8224	nmod	NaCl,Tween
R2527	T8235	T8224	punct	/,Tween
R2528	T8236	T8237	nummod	0.1,%
R2529	T8237	T8224	compound	%,Tween
R253	T2625	T2626	advmod	in,vivo
R2530	T8238	T8224	punct	-,Tween
R2531	T8239	T8224	nummod	20,Tween
R2532	T8240	T8224	punct	),Tween
R2533	T8241	T8212	prep	for,incubated
R2534	T8242	T8243	nummod	1,h
R2535	T8243	T8241	pobj	h,for
R2536	T8244	T8212	prep	at,incubated
R2537	T8245	T8246	compound	room,temperature
R2538	T8246	T8244	pobj	temperature,at
R2539	T8247	T8212	prep	before,incubated
R254	T2626	T2616	advmod	vivo,tested
R2540	T8248	T8247	pcomp	probing,before
R2541	T8249	T8248	prep	with,probing
R2542	T8250	T8251	amod	primary,antibodies
R2543	T8251	T8249	pobj	antibodies,with
R2544	T8252	T8251	prep	against,antibodies
R2545	T8253	T8252	pobj	p53,against
R2546	T8254	T8255	punct	(,Novacastra
R2547	T8255	T8253	parataxis	Novacastra,p53
R2548	T8256	T8255	dep	CM,Novacastra
R2549	T8257	T8256	punct	-,CM
R255	T2627	T2616	prep	by,tested
R2550	T8258	T8256	nummod	5,CM
R2551	T8259	T8255	punct	", ",Novacastra
R2552	T8260	T8255	punct	),Novacastra
R2553	T8261	T8253	cc	and,p53
R2554	T8262	T8263	punct	-,actin
R2555	T8263	T8253	conj	actin,p53
R2556	T8264	T8265	punct	(,Sigma
R2557	T8265	T8263	parataxis	Sigma,actin
R2558	T8266	T8265	punct	),Sigma
R2559	T8267	T8212	punct	.,incubated
R256	T2628	T2627	pcomp	targeting,by
R2560	T8269	T8270	amod	Secondary,antibodies
R2561	T8270	T8271	nsubj	antibodies,include
R2562	T8272	T8270	acl	used,antibodies
R2563	T8273	T8274	npadvmod	peroxidase,conjugated
R2564	T8274	T8276	amod	conjugated,IgG
R2565	T8275	T8274	punct	-,conjugated
R2566	T8276	T8271	dobj	IgG,include
R2567	T8277	T8276	compound	goat,IgG
R2568	T8278	T8276	compound	anti-mouse,IgG
R2569	T8279	T8276	cc	and,IgG
R257	T2629	T2630	amod	subtle,mutations
R2570	T8280	T8281	compound	anti-rabbit,IgG
R2571	T8281	T8276	conj	IgG,IgG
R2572	T8282	T8283	punct	(,Pierce
R2573	T8283	T8281	parataxis	Pierce,IgG
R2574	T8284	T8283	punct	),Pierce
R2575	T8285	T8271	punct	.,include
R2576	T8287	T8288	amod	Probed,blots
R2577	T8288	T8289	nsubjpass	blots,incubated
R2578	T8290	T8289	auxpass	were,incubated
R2579	T8291	T8289	prep	with,incubated
R258	T2630	T2628	dobj	mutations,targeting
R2580	T8292	T8293	nmod	Pierce,substrate
R2581	T8293	T8291	pobj	substrate,with
R2582	T8294	T8295	nmod	Supersignal,West
R2583	T8295	T8293	nmod	West,substrate
R2584	T8296	T8293	nmod	Pico,substrate
R2585	T8297	T8293	amod	chemiluminescent,substrate
R2586	T8298	T8289	cc	and,incubated
R2587	T8299	T8289	conj	exposed,incubated
R2588	T8300	T8299	prep	to,exposed
R2589	T8301	T8302	compound	X-ray,films
R259	T2631	T2628	prep	at,targeting
R2590	T8302	T8300	pobj	films,to
R2591	T8303	T8289	punct	.,incubated
R2592	T8467	T8468	compound	Flow,cytometry
R2593	T8470	T8471	compound	Log,cells
R2594	T8471	T8473	nsubjpass	cells,irradiated
R2595	T8472	T8471	compound	phase,cells
R2596	T8474	T8473	auxpass	were,irradiated
R2597	T8475	T8473	prep	at,irradiated
R2598	T8476	T8475	pobj	RT,at
R2599	T8477	T8473	prep	with,irradiated
R26	T467	T461	aux	has,gained
R260	T2632	T2633	det	the,locus
R2600	T8478	T8479	det	a,irradiator
R2601	T8479	T8477	pobj	irradiator,with
R2602	T8480	T8481	nummod	60,γ
R2603	T8481	T8479	nmod	γ,irradiator
R2604	T8482	T8481	nmod	Co,γ
R2605	T8483	T8481	punct	-,γ
R2606	T8484	T8473	prep	at,irradiated
R2607	T8485	T8484	pobj	doses,at
R2608	T8486	T8485	prep	of,doses
R2609	T8487	T8488	nummod	6,Gy
R261	T2633	T2631	pobj	locus,at
R2610	T8488	T8486	pobj	Gy,of
R2611	T8489	T8487	cc	or,6
R2612	T8490	T8487	conj	12,6
R2613	T8491	T8473	cc	and,irradiated
R2614	T8492	T8473	conj	incubated,irradiated
R2615	T8493	T8492	prep	for,incubated
R2616	T8494	T8495	nummod	24,h
R2617	T8495	T8493	pobj	h,for
R2618	T8496	T8473	punct	.,irradiated
R2619	T8498	T8499	nsubjpass	Cells,labeled
R262	T2695	T2690	advmod	vivo,from
R2620	T8500	T8499	auxpass	were,labeled
R2621	T8501	T8499	advmod	then,labeled
R2622	T8502	T8499	dep	pulse,labeled
R2623	T8503	T8499	punct	-,labeled
R2624	T8504	T8499	prep	for,labeled
R2625	T8505	T8506	nummod	1,h
R2626	T8506	T8504	pobj	h,for
R2627	T8507	T8499	prep	with,labeled
R2628	T8508	T8507	pobj	BrdU,with
R2629	T8509	T8510	punct	(,μM
R263	T2634	T2633	compound	p53,locus
R2630	T8510	T8508	parataxis	μM,BrdU
R2631	T8511	T8510	nummod	10,μM
R2632	T8512	T8510	punct	),μM
R2633	T8513	T8499	punct	", ",labeled
R2634	T8514	T8499	dep	fixed,labeled
R2635	T8515	T8514	prep	in,fixed
R2636	T8516	T8517	nummod	70,%
R2637	T8517	T8518	compound	%,ethanol
R2638	T8518	T8515	pobj	ethanol,in
R2639	T8519	T8499	punct	", ",labeled
R264	T2696	T2689	cc	and,phenotypes
R2640	T8520	T8521	amod	double,stained
R2641	T8521	T8499	dep	stained,labeled
R2642	T8522	T8521	punct	-,stained
R2643	T8523	T8521	prep	with,stained
R2644	T8524	T8525	compound	FITC,anti-BrdU
R2645	T8525	T8523	pobj	anti-BrdU,with
R2646	T8526	T8525	cc	and,anti-BrdU
R2647	T8527	T8528	compound	propidium,iodide
R2648	T8528	T8525	conj	iodide,anti-BrdU
R2649	T8529	T8499	punct	", ",labeled
R265	T2635	T2628	advcl	using,targeting
R2650	T8530	T8531	advmod	then,sorted
R2651	T8531	T8499	dep	sorted,labeled
R2652	T8532	T8531	prep	by,sorted
R2653	T8533	T8532	pcomp	using,by
R2654	T8534	T8535	det	a,machine
R2655	T8535	T8533	dobj	machine,using
R2656	T8536	T8537	compound	Becton,Dickinson
R2657	T8537	T8535	compound	Dickinson,machine
R2658	T8538	T8535	compound	FACScan,machine
R2659	T8539	T8499	punct	.,labeled
R266	T2697	T2698	compound	transfection,data
R2660	T8541	T8542	nsubjpass	Data,analyzed
R2661	T8543	T8542	auxpass	were,analyzed
R2662	T8544	T8542	advcl	using,analyzed
R2663	T8545	T8546	compound	Becton,Dickinson
R2664	T8546	T8547	compound	Dickinson,Pro
R2665	T8547	T8544	dobj	Pro,using
R2666	T8548	T8547	compound	Cellquest,Pro
R2667	T8549	T8542	punct	.,analyzed
R267	T2698	T2689	conj	data,phenotypes
R2672	T11673	T11674	det	The,rationale
R2673	T11674	T11675	nsubjpass	rationale,detailed
R2674	T11676	T11674	prep	for,rationale
R2675	T11677	T11678	compound	RMCE,ASAP
R2676	T11678	T11676	pobj	ASAP,for
R2677	T11679	T11678	punct	-,ASAP
R2678	T11680	T11675	auxpass	is,detailed
R2679	T11681	T11675	prep	in,detailed
R268	T2636	T2637	amod	homologous,recombination
R2680	T11682	T11681	pobj	Figure,in
R2681	T11683	T11682	nummod	1,Figure
R2682	T11684	T11675	punct	.,detailed
R2683	T11686	T11687	det	The,step
R2684	T11687	T11689	nsubj	step,requires
R2685	T11688	T11687	amod	first,step
R2686	T11690	T11689	xcomp	generating,requires
R2687	T11691	T11692	det	a,allele
R2688	T11692	T11690	dobj	allele,generating
R2689	T11693	T11692	amod	floxed,allele
R269	T2699	T2685	auxpass	were,observed
R2690	T11694	T11690	prep	in,generating
R2691	T11695	T11696	compound	ES,cells
R2692	T11696	T11694	pobj	cells,in
R2693	T11697	T11698	dep	that,serve
R2694	T11698	T11696	relcl	serve,cells
R2695	T11699	T11698	aux	will,serve
R2696	T11700	T11698	prep	as,serve
R2697	T11701	T11702	det	the,substrate
R2698	T11702	T11700	pobj	substrate,as
R2699	T11703	T11702	prep	for,substrate
R27	T468	T461	auxpass	been,gained
R270	T2637	T2635	dobj	recombination,using
R2700	T11704	T11705	amod	subsequent,exchanges
R2701	T11705	T11703	pobj	exchanges,for
R2702	T11706	T11707	punct	(,Figure
R2703	T11707	T11689	parataxis	Figure,requires
R2704	T11708	T11709	npadvmod	RMCE,ready
R2705	T11709	T11711	amod	ready,cell
R2706	T11710	T11709	punct	-,ready
R2707	T11711	T11707	dep	cell,Figure
R2708	T11712	T11711	compound	ES,cell
R2709	T11713	T11707	punct	", ",Figure
R271	T2701	T2702	punct	[,1
R2710	T11714	T11707	nummod	1,Figure
R2711	T11715	T11707	punct	),Figure
R2712	T11716	T11689	punct	.,requires
R2713	T11718	T11719	det	The,strategy
R2714	T11719	T11721	nsubjpass	strategy,detailed
R2715	T11720	T11719	amod	targeting,strategy
R2716	T11722	T11721	auxpass	is,detailed
R2717	T11723	T11721	prep	in,detailed
R2718	T11724	T11723	pobj	Figure,in
R2719	T11725	T11724	nummod	2,Figure
R272	T2702	T2685	parataxis	1,observed
R2720	T11726	T11721	punct	.,detailed
R2721	T11728	T11729	det	The,frequency
R2722	T11729	T11730	nsubj	frequency,was
R2723	T11731	T11729	prep	of,frequency
R2724	T11732	T11731	pobj	targeting,of
R2725	T11733	T11734	nummod	4,%
R2726	T11734	T11730	attr	%,was
R2727	T11735	T11736	punct	(,Figure
R2728	T11736	T11730	parataxis	Figure,was
R2729	T11737	T11738	compound	12,300
R273	T2703	T2702	advmod	e.g.,1
R2730	T11738	T11740	nummod	300,clones
R2731	T11739	T11738	punct	/,300
R2732	T11740	T11736	dep	clones,Figure
R2733	T11741	T11742	npadvmod	puromycin,resistant
R2734	T11742	T11740	amod	resistant,clones
R2735	T11743	T11742	punct	-,resistant
R2736	T11850	T11845	punct	-,furanosyl
R2737	T11851	T11845	nummod	2,furanosyl
R2738	T11852	T11845	punct	-,furanosyl
R2739	T11853	T11845	nmod	fluoro,furanosyl
R274	T2638	T2635	prep	in,using
R2740	T11854	T11845	punct	-,furanosyl
R2741	T11855	T11845	nummod	1,furanosyl
R2742	T11856	T11845	punct	-,furanosyl
R2743	T11857	T11845	compound	b,furanosyl
R2744	T11858	T11845	punct	-,furanosyl
R2745	T11859	T11845	compound	d,furanosyl
R2746	T11860	T11845	punct	-,furanosyl
R2747	T11861	T11845	compound	arabino,furanosyl
R2748	T11862	T11845	punct	-,furanosyl
R2749	T11863	T11845	punct	),furanosyl
R275	T2704	T2702	nmod	Refs,1
R2750	T11864	T11842	punct	-,5
R2751	T11744	T11740	punct	", ",clones
R2752	T11865	T11840	pobj	iodouracil,to
R2753	T11745	T11740	acl	analyzed,clones
R2754	T11746	T11745	prep	by,analyzed
R2755	T11747	T11748	compound	Southern,blot
R2756	T11866	T11865	punct	-,iodouracil
R2757	T11748	T11746	pobj	blot,by
R2758	T11749	T11748	cc	and,blot
R2759	T11750	T11751	amod	long,range
R276	T2705	T2702	punct	(,1
R2760	T11867	T11865	punct	(,iodouracil
R2761	T11751	T11753	compound	range,PCR
R2762	T11752	T11751	punct	-,range
R2763	T11753	T11748	conj	PCR,blot
R2764	T11868	T11865	appos	FIAU,iodouracil
R2765	T11754	T11736	punct	", ",Figure
R2766	T11755	T11736	nummod	2,Figure
R2767	T11756	T11736	punct	),Figure
R2768	T11869	T11839	punct	),resistant
R2769	T11757	T11730	punct	.,was
R277	T2639	T2640	amod	embryonic,stem
R2770	T11759	T11760	nsubj	We,tested
R2771	T11761	T11760	advmod	next,tested
R2772	T11762	T11763	det	the,efficiency
R2773	T11870	T11839	prep	due,resistant
R2774	T11763	T11760	dobj	efficiency,tested
R2775	T11764	T11763	prep	of,efficiency
R2776	T11765	T11764	pobj	RMCE,of
R2777	T11871	T11870	pcomp	to,due
R2778	T11766	T11763	prep	in,efficiency
R2779	T11767	T11768	compound	ES,cells
R278	T2706	T2707	punct	–,5
R2780	T11768	T11766	pobj	cells,in
R2781	T11872	T11873	compound	TK,loss
R2782	T11769	T11760	punct	", ",tested
R2783	T11873	T11870	pobj	loss,due
R2784	T11874	T11834	cc	and,picked
R2785	T11770	T11760	advcl	using,tested
R2786	T11771	T11772	det	a,construct
R2787	T11772	T11770	dobj	construct,using
R2788	T11875	T11834	conj	analyzed,picked
R2789	T11773	T11772	compound	replacement,construct
R279	T2707	T2702	prep	5,1
R2790	T11774	T11772	acl	encoding,construct
R2791	T11876	T11877	poss	their,DNA
R2792	T11775	T11774	dobj	p53,encoding
R2793	T11877	T11875	dobj	DNA,analyzed
R2794	T11776	T11772	acl	fused,construct
R2795	T11777	T11776	prep	to,fused
R2796	T11778	T11777	pobj	GFP,to
R2797	T11878	T11875	prep	by,analyzed
R2798	T11779	T11778	punct	(,GFP
R2799	T11780	T11778	appos	p53GFP,GFP
R28	T469	T461	prep	on,gained
R280	T2708	T2702	punct	),1
R2800	T11781	T11770	punct	),using
R2801	T11879	T11878	pobj	PCR,by
R2802	T11782	T11783	aux	to,enable
R2803	T11783	T11770	advcl	enable,using
R2804	T11784	T11785	amod	tracking,p53
R2805	T11880	T11879	cc	and,PCR
R2806	T11785	T11783	dobj	p53,enable
R2807	T11786	T11783	prep	in,enable
R2808	T11787	T11788	amod	individual,cells
R2809	T11881	T11882	compound	Southern,blot
R281	T2640	T2641	nmod	stem,cells
R2810	T11788	T11786	pobj	cells,in
R2811	T11789	T11788	amod	live,cells
R2812	T11790	T11760	punct	.,tested
R2813	T11882	T11879	conj	blot,PCR
R2814	T11792	T11793	advmod	Importantly,used
R2815	T11883	T11834	punct	.,picked
R2816	T11794	T11793	advmod	however,used
R2817	T11795	T11793	punct	", ",used
R2818	T11796	T11797	compound	GFP,fluorescence
R2819	T11885	T11886	advmod	Strikingly,identified
R282	T2709	T2702	punct	],1
R2820	T11797	T11793	nsubjpass	fluorescence,used
R2821	T11798	T11793	auxpass	was,used
R2822	T11799	T11793	neg	not,used
R2823	T11800	T11801	aux	to,screen
R2824	T11801	T11793	advcl	screen,used
R2825	T11802	T11801	dobj	cells,screen
R2826	T11803	T11802	prep	with,cells
R2827	T11887	T11886	punct	", ",identified
R2828	T11804	T11805	amod	targeted,events
R2829	T11805	T11803	pobj	events,with
R283	T2641	T2638	pobj	cells,in
R2830	T11806	T11793	punct	", ",used
R2831	T11807	T11808	mark	as,wanted
R2832	T11888	T11889	nummod	54,recombinants
R2833	T11808	T11793	advcl	wanted,used
R2834	T11809	T11808	nsubj	we,wanted
R2835	T11810	T11811	aux	to,develop
R2836	T11811	T11808	xcomp	develop,wanted
R2837	T11889	T11886	nsubjpass	recombinants,identified
R2838	T11812	T11813	det	a,method
R2839	T11813	T11811	dobj	method,develop
R284	T2710	T2700	punct	", ",need
R2840	T11814	T11813	amod	general,method
R2841	T11890	T11889	amod	proper,recombinants
R2842	T11815	T11816	aux	to,isolate
R2843	T11816	T11811	advcl	isolate,develop
R2844	T11891	T11886	auxpass	were,identified
R2845	T11817	T11818	npadvmod	marker,free
R2846	T11818	T11820	amod	free,recombinants
R2847	T11819	T11818	punct	-,free
R2848	T11892	T11886	punct	", ",identified
R2849	T11820	T11816	dobj	recombinants,isolate
R285	T2642	T2640	punct	(,stem
R2850	T11821	T11793	punct	.,used
R2851	T11893	T11886	advcl	indicating,identified
R2852	T11823	T11824	det	The,strategy
R2853	T11824	T11826	nsubjpass	strategy,detailed
R2854	T11825	T11824	compound	exchange,strategy
R2855	T11894	T11895	advmod	very,high
R2856	T11827	T11826	auxpass	is,detailed
R2857	T11895	T11896	amod	high,efficiency
R2858	T11828	T11826	prep	in,detailed
R2859	T11829	T11830	compound	Figure,3A
R286	T2711	T2712	advmod	more,targeted
R2860	T11830	T11828	pobj	3A,in
R2861	T11896	T11893	dobj	efficiency,indicating
R2862	T11831	T11826	punct	.,detailed
R2863	T11833	T11834	nsubj	We,picked
R2864	T11897	T11896	compound	RMCE,efficiency
R2865	T11835	T11836	nummod	65,clones
R2866	T11836	T11834	dobj	clones,picked
R2867	T11898	T11899	punct	(,%
R2868	T11837	T11838	compound	ES,cell
R2869	T11838	T11836	compound	cell,clones
R287	T2712	T2713	amod	targeted,mutations
R2870	T11839	T11836	relcl	resistant,clones
R2871	T11899	T11896	parataxis	%,efficiency
R2872	T11840	T11839	prep	to,resistant
R2873	T11841	T11842	nummod	1,5
R2874	T11900	T11899	nummod	83,%
R2875	T11842	T11865	nummod	5,iodouracil
R2876	T11843	T11842	punct	-,5
R2877	T11901	T11899	punct	),%
R2878	T11844	T11845	punct	(,furanosyl
R2879	T11845	T11842	parataxis	furanosyl,5
R288	T2643	T2640	appos	ES,stem
R2880	T11846	T11845	punct	-,furanosyl
R2881	T11902	T11886	punct	.,identified
R2882	T11847	T11845	nummod	2,furanosyl
R2883	T11848	T11845	punct	-,furanosyl
R2884	T11849	T11845	nmod	deoxy,furanosyl
R2885	T11904	T11905	nsubj	RMCE,proved
R2886	T11906	T11905	advmod	also,proved
R2887	T11907	T11908	aux	to,be
R2888	T11908	T11905	xcomp	be,proved
R2889	T11909	T11908	acomp	precise,be
R289	T2713	T2700	nsubj	mutations,need
R2890	T11910	T11908	punct	", ",be
R2891	T11955	T11954	amod	expected,size
R2892	T11956	T11954	punct	(,size
R2893	T11911	T11912	mark	as,detected
R2894	T11957	T11958	quantmod	ca.,80
R2895	T11958	T11959	nummod	80,kDa
R2896	T11912	T11908	advcl	detected,be
R2897	T11959	T11954	appos	kDa,size
R2898	T11960	T11934	punct	),analyzed
R2899	T11961	T11934	punct	.,analyzed
R29	T470	T471	compound	p53,regulation
R290	T2714	T2715	aux	to,generated
R2900	T11913	T11914	det	no,bands
R2901	T11963	T11964	advmod	Surprisingly,altered
R2902	T11914	T11912	nsubjpass	bands,detected
R2903	T11964	T11973	ccomp	altered,were
R2904	T11965	T11964	punct	", ",altered
R2905	T11966	T11967	det	the,fusion
R2906	T11915	T11914	amod	aberrant,bands
R2907	T11967	T11964	nsubj	fusion,altered
R2908	T11968	T11967	prep	of,fusion
R2909	T11916	T11912	auxpass	were,detected
R291	T2715	T2700	xcomp	generated,need
R2910	T11969	T11968	pobj	GFP,of
R2911	T11970	T11967	prep	to,fusion
R2912	T11971	T11970	pobj	p53,to
R2913	T11917	T11912	prep	in,detected
R2914	T11972	T11964	advmod	apparently,altered
R2915	T11974	T11975	compound	p53,stability
R2916	T11918	T11917	pobj	PCR,in
R2917	T11975	T11964	dobj	stability,altered
R2918	T11976	T11973	punct	: ,were
R2919	T11977	T11978	amod	steady,state
R292	T2716	T2715	auxpass	be,generated
R2920	T11919	T11918	cc	and,PCR
R2921	T11978	T11980	compound	state,levels
R2922	T11979	T11978	punct	-,state
R2923	T11980	T11973	nsubj	levels,were
R2924	T11920	T11921	compound	Southern,blots
R2925	T11981	T11980	prep	of,levels
R2926	T11982	T11981	pobj	p53GFP,of
R2927	T11983	T11984	advmod	much,higher
R2928	T11984	T11973	acomp	higher,were
R2929	T11985	T11984	prep	than,higher
R293	T2644	T2641	punct	),cells
R2930	T11986	T11985	pobj	those,than
R2931	T11987	T11986	prep	of,those
R2932	T11921	T11918	conj	blots,PCR
R2933	T11988	T11989	amod	wild,type
R2934	T11989	T11991	compound	type,p53
R2935	T11990	T11989	punct	-,type
R2936	T11922	T11923	punct	(,3A
R2937	T11991	T11987	pobj	p53,of
R2938	T11992	T11991	punct	(,p53
R2939	T11923	T11912	parataxis	3A,detected
R294	T2717	T2715	cc	and,generated
R2940	T11993	T11991	appos	p53WT,p53
R2941	T11994	T11991	punct	),p53
R2942	T11995	T11991	prep	in,p53
R2943	T11924	T11923	compound	Figure,3A
R2944	T11996	T11997	amod	unstressed,cells
R2945	T11997	T11995	pobj	cells,in
R2946	T11998	T11973	punct	", ",were
R2947	T11925	T11923	punct	),3A
R2948	T11999	T11973	cc	and,were
R2949	T12000	T12001	aux	did,vary
R295	T2718	T2715	conj	analyzed,generated
R2950	T12001	T11973	conj	vary,were
R2951	T11926	T11905	punct	.,proved
R2952	T12002	T12001	neg	not,vary
R2953	T12003	T12001	advmod	significantly,vary
R2954	T11928	T11929	nmod	p53,GFP
R2955	T12004	T12001	prep	after,vary
R2956	T12005	T12006	compound	DNA,damage
R2957	T12006	T12004	pobj	damage,after
R2958	T12007	T12001	punct	", ",vary
R2959	T12008	T12009	mark	so,were
R296	T2645	T2646	aux	to,generate
R2960	T11929	T11932	compound	GFP,clones
R2961	T12009	T12001	advcl	were,vary
R2962	T12010	T12009	mark	that,were
R2963	T12011	T12012	det	the,levels
R2964	T11930	T11929	punct	+,GFP
R2965	T12012	T12009	nsubj	levels,were
R2966	T12013	T12012	prep	for,levels
R2967	T12014	T12015	preconj	both,p53WT
R2968	T12015	T12013	pobj	p53WT,for
R2969	T11931	T11929	punct	/,GFP
R297	T2719	T2718	prep	in,analyzed
R2970	T12016	T12015	cc	and,p53WT
R2971	T12017	T12015	conj	p53GFP,p53WT
R2972	T12018	T12009	acomp	similar,were
R2973	T11932	T11934	nsubjpass	clones,analyzed
R2974	T12019	T12009	prep	after,were
R2975	T12020	T12021	compound	adriamycin,treatment
R2976	T12021	T12019	pobj	treatment,after
R2977	T11933	T11932	compound	ES,clones
R2978	T12022	T12023	punct	(,3A
R2979	T12023	T12009	parataxis	3A,were
R298	T2720	T2721	amod	multiple,tissues
R2980	T12024	T12023	compound	Figure,3A
R2981	T11935	T11934	auxpass	were,analyzed
R2982	T12025	T12023	punct	),3A
R2983	T12026	T11973	punct	.,were
R2984	T12028	T12029	nummod	Six,clones
R2985	T11936	T11934	prep	by,analyzed
R2986	T12029	T12035	nsubjpass	clones,injected
R2987	T12030	T12029	amod	independent,clones
R2988	T11937	T11938	compound	western,blot
R2989	T12031	T12032	nmod	p53,GFP
R299	T2721	T2719	pobj	tissues,in
R2990	T12032	T12029	compound	GFP,clones
R2991	T12033	T12032	punct	+,GFP
R2992	T11938	T11936	pobj	blot,by
R2993	T12034	T12032	punct	/,GFP
R2994	T12036	T12035	auxpass	were,injected
R2995	T11939	T11934	prep	with,analyzed
R2996	T12037	T12035	prep	into,injected
R2997	T12038	T12037	pobj	blastocysts,into
R2998	T12039	T12035	cc	and,injected
R2999	T11940	T11941	det	an,antibody
R30	T471	T469	pobj	regulation,on
R300	T2722	T2723	aux	to,formulate
R3000	T12040	T12035	conj	transferred,injected
R3001	T12041	T12040	prep	to,transferred
R3002	T12042	T12043	amod	pseudo,pregnant
R3003	T11941	T11939	pobj	antibody,with
R3004	T12043	T12045	amod	pregnant,females
R3005	T12044	T12043	punct	-,pregnant
R3006	T11942	T11941	prep	against,antibody
R3007	T12045	T12041	pobj	females,to
R3008	T12046	T12040	advcl	using,transferred
R3009	T12047	T12048	amod	standard,procedures
R301	T2723	T2718	advcl	formulate,analyzed
R3010	T11943	T11942	pobj	p53,against
R3011	T12048	T12046	dobj	procedures,using
R3012	T12049	T12035	punct	.,injected
R3013	T11944	T11934	punct	", ",analyzed
R3014	T12051	T12052	advmod	Strikingly,obtained
R3015	T12053	T12052	punct	", ",obtained
R3016	T11945	T11934	cc	and,analyzed
R3017	T12054	T12055	det	no,pregnancies
R3018	T12055	T12052	nsubjpass	pregnancies,obtained
R3019	T12056	T12052	auxpass	were,obtained
R302	T2724	T2725	advmod	more,accurate
R3020	T11946	T11934	conj	found,analyzed
R3021	T12057	T12052	punct	.,obtained
R3022	T12059	T12060	nsubjpass	It,shown
R3023	T11947	T11948	aux	to,express
R3024	T12060	T12063	ccomp	shown,contain
R3025	T11948	T11946	xcomp	express,found
R3026	T11949	T11950	det	an,band
R3027	T12061	T12060	aux	has,shown
R3028	T11950	T11948	dobj	band,express
R3029	T12062	T12060	auxpass	been,shown
R303	T2646	T2635	advcl	generate,using
R3030	T12064	T12065	mark	that,regulated
R3031	T12065	T12060	ccomp	regulated,shown
R3032	T12066	T12067	det	the,pathway
R3033	T12067	T12065	nsubjpass	pathway,regulated
R3034	T11951	T11950	amod	additional,band
R3035	T12068	T12067	compound	p53,pathway
R3036	T12069	T12065	auxpass	is,regulated
R3037	T12070	T12071	advmod	very,differently
R3038	T12071	T12065	advmod	differently,regulated
R3039	T11952	T11948	prep	at,express
R304	T2725	T2726	amod	accurate,models
R3040	T12072	T12065	prep	in,regulated
R3041	T12073	T12074	nmod	ES,cells
R3042	T12074	T12072	pobj	cells,in
R3043	T11953	T11954	det	the,size
R3044	T12075	T12073	cc	and,ES
R3045	T12076	T12073	conj	somatic,ES
R3046	T11954	T11952	pobj	size,at
R3047	T12077	T12063	punct	: ,contain
R3048	T12078	T12079	compound	ES,cells
R3049	T12079	T12063	nsubj	cells,contain
R305	T2647	T2648	amod	mutant,mice
R3050	T12167	T12168	det	this,possibility
R3051	T12080	T12081	advmod	relatively,high
R3052	T12081	T12082	amod	high,levels
R3053	T12082	T12063	dobj	levels,contain
R3054	T12083	T12082	compound	p53,levels
R3055	T12084	T12063	cc	and,contain
R3056	T12168	T12165	dobj	possibility,test
R3057	T12085	T12063	conj	lack,contain
R3058	T12086	T12087	det	the,responses
R3059	T12087	T12085	dobj	responses,lack
R306	T2726	T2723	dobj	models,formulate
R3060	T12169	T12166	punct	", ",performed
R3061	T12088	T12087	nmod	p53,responses
R3062	T12089	T12088	punct	-,p53
R3063	T12090	T12088	amod	mediated,p53
R3064	T12170	T12166	nsubj	we,performed
R3065	T12091	T12092	compound	DNA,damage
R3066	T12092	T12087	compound	damage,responses
R3067	T12171	T12166	dobj	RMCE,performed
R3068	T12093	T12087	acl	found,responses
R3069	T12094	T12093	prep	in,found
R307	T2727	T2726	prep	of,models
R3070	T12095	T12096	amod	somatic,cells
R3071	T12172	T12166	prep	with,performed
R3072	T12096	T12094	pobj	cells,in
R3073	T12097	T12098	punct	(,18
R3074	T12098	T12085	parataxis	18,lack
R3075	T12099	T12098	punct	),18
R3076	T12173	T12174	det	a,gene
R3077	T12100	T12063	punct	.,contain
R3078	T12174	T12172	pobj	gene,with
R3079	T12102	T12103	nsubj	This,suggested
R308	T2728	T2729	compound	p53,regulation
R3080	T12175	T12176	compound	p53,fusion
R3081	T12103	T12120	ccomp	suggested,speculate
R3082	T12104	T12103	punct	", ",suggested
R3083	T12105	T12103	advmod	together,suggested
R3084	T12106	T12105	prep	with,together
R3085	T12107	T12108	det	the,observation
R3086	T12108	T12106	pobj	observation,with
R3087	T12176	T12174	compound	fusion,gene
R3088	T12109	T12110	mark	that,decrease
R3089	T12110	T12108	acl	decrease,observation
R309	T2729	T2727	pobj	regulation,of
R3090	T12111	T12112	compound	p53,levels
R3091	T12177	T12178	prep	in,deleted
R3092	T12112	T12110	nsubj	levels,decrease
R3093	T12113	T12110	prep	during,decrease
R3094	T12114	T12115	compound	mouse,embryogenesis
R3095	T12178	T12174	advcl	deleted,gene
R3096	T12115	T12113	pobj	embryogenesis,during
R3097	T12116	T12117	punct	(,19
R3098	T12179	T12177	pobj	which,in
R3099	T12117	T12110	parataxis	19,decrease
R31	T472	T461	prep	by,gained
R310	T2730	T2700	punct	.,need
R3100	T12118	T12117	punct	),19
R3101	T12119	T12103	punct	", ",suggested
R3102	T12180	T12181	det	the,domain
R3103	T12121	T12122	det	an,explanation
R3104	T12181	T12178	nsubjpass	domain,deleted
R3105	T12122	T12103	dobj	explanation,suggested
R3106	T12123	T12122	prep	for,explanation
R3107	T12124	T12125	det	the,lack
R3108	T12182	T12181	nmod	p53,domain
R3109	T12125	T12123	pobj	lack,for
R311	T2648	T2646	dobj	mice,generate
R3110	T12126	T12125	amod	observed,lack
R3111	T12127	T12125	prep	of,lack
R3112	T12183	T12184	npadvmod	proline,rich
R3113	T12128	T12127	pobj	pregnancies,of
R3114	T12129	T12120	punct	: ,speculate
R3115	T12130	T12120	nsubj	we,speculate
R3116	T12184	T12181	amod	rich,domain
R3117	T12131	T12132	mark	that,prevented
R3118	T12132	T12120	ccomp	prevented,speculate
R3119	T12185	T12184	punct	-,rich
R312	T2732	T2733	advmod	However,is
R3120	T12133	T12134	det	the,levels
R3121	T12134	T12132	nsubj	levels,prevented
R3122	T12135	T12134	amod	high,levels
R3123	T12186	T12181	punct	(,domain
R3124	T12136	T12134	prep	of,levels
R3125	T12137	T12136	pobj	p53GFP,of
R3126	T12187	T12181	appos	PRD,domain
R3127	T12138	T12134	prep	in,levels
R3128	T12139	T12140	det	the,cells
R3129	T12140	T12138	pobj	cells,in
R313	T2649	T2616	punct	.,tested
R3130	T12141	T12140	compound	ES,cells
R3131	T12188	T12178	punct	),deleted
R3132	T12142	T12140	acl	injected,cells
R3133	T12143	T12142	prep	into,injected
R3134	T12144	T12143	pobj	blastocysts,into
R3135	T12145	T12132	aux	might,prevented
R3136	T12146	T12132	aux	have,prevented
R3137	T12189	T12178	auxpass	was,deleted
R3138	T12147	T12148	amod	normal,development
R3139	T12148	T12132	dobj	development,prevented
R314	T2734	T2733	punct	", ",is
R3140	T12149	T12148	amod	embryonic,development
R3141	T12190	T12191	punct	(,p53ΔP
R3142	T12150	T12151	prep	once,began
R3143	T12151	T12132	advcl	began,prevented
R3144	T12152	T12153	det	these,cells
R3145	T12191	T12178	parataxis	p53ΔP,deleted
R3146	T12153	T12151	nsubj	cells,began
R3147	T12154	T12155	aux	to,differentiate
R3148	T12155	T12151	xcomp	differentiate,began
R3149	T12192	T12191	punct	),p53ΔP
R315	T2735	T2733	csubj	using,is
R3150	T12156	T12151	cc	and,began
R3151	T12157	T12158	det	the,pathway
R3152	T12193	T12178	punct	", ",deleted
R3153	T12158	T12160	nsubj	pathway,became
R3154	T12159	T12158	compound	p53,pathway
R3155	T12160	T12151	conj	became,began
R3156	T12194	T12178	cc	and,deleted
R3157	T12161	T12160	acomp	functional,became
R3158	T12162	T12120	punct	.,speculate
R3159	T12195	T12196	det	the,p53ΔP
R316	T2651	T2652	det	The,strength
R3160	T12164	T12165	aux	To,test
R3161	T12165	T12166	advcl	test,performed
R3162	T12196	T12197	nsubjpass	p53ΔP,fused
R3163	T12197	T12178	conj	fused,deleted
R3164	T12198	T12197	auxpass	was,fused
R3165	T12273	T12272	compound	ES,cells
R3166	T12274	T12275	punct	(,3B
R3167	T12199	T12197	prep	to,fused
R3168	T12275	T12257	parataxis	3B,revealed
R3169	T12276	T12275	compound	Figure,3B
R317	T2736	T2737	amod	homologous,recombination
R3170	T12277	T12275	punct	),3B
R3171	T12200	T12199	pobj	GFP,to
R3172	T12278	T12257	punct	.,revealed
R3173	T12201	T12166	punct	.,performed
R3174	T12280	T12281	mark	As,expected
R3175	T12203	T12204	nsubj	We,used
R3176	T12281	T12282	advcl	expected,correlated
R3177	T12282	T12287	ccomp	correlated,was
R3178	T12283	T12282	punct	", ",correlated
R3179	T12205	T12206	det	this,mutant
R318	T2652	T2653	nsubj	strength,is
R3180	T12284	T12285	det	the,deletion
R3181	T12285	T12282	nsubj	deletion,correlated
R3182	T12286	T12285	compound	PRD,deletion
R3183	T12206	T12204	dobj	mutant,used
R3184	T12288	T12282	prep	with,correlated
R3185	T12289	T12290	amod	lower,levels
R3186	T12290	T12288	pobj	levels,with
R3187	T12291	T12290	compound	expression,levels
R3188	T12292	T12287	punct	: ,was
R3189	T12207	T12208	mark	because,decreases
R319	T2737	T2735	dobj	recombination,using
R3190	T12293	T12287	nsubj	p53ΔPGFP,was
R3191	T12294	T12295	advmod	much,less
R3192	T12295	T12296	advmod	less,abundant
R3193	T12208	T12204	advcl	decreases,used
R3194	T12296	T12287	acomp	abundant,was
R3195	T12297	T12296	prep	than,abundant
R3196	T12298	T12297	pobj	p53WT,than
R3197	T12209	T12208	csubj	deleting,decreases
R3198	T12299	T12298	prep	in,p53WT
R3199	T12300	T12301	det	all,clones
R32	T473	T472	pcomp	targeting,by
R320	T2738	T2735	prep	in,using
R3200	T12301	T12299	pobj	clones,in
R3201	T12210	T12211	det	the,domain
R3202	T12302	T12303	nmod	p53,ΔPGFP
R3203	T12303	T12301	compound	ΔPGFP,clones
R3204	T12304	T12303	punct	+,ΔPGFP
R3205	T12211	T12209	dobj	domain,deleting
R3206	T12305	T12303	punct	/,ΔPGFP
R3207	T12306	T12307	punct	(,3B
R3208	T12307	T12287	parataxis	3B,was
R3209	T12212	T12213	npadvmod	proline,rich
R321	T2654	T2652	prep	of,strength
R3210	T12308	T12307	compound	Figure,3B
R3211	T12309	T12307	punct	),3B
R3212	T12310	T12287	punct	.,was
R3213	T12213	T12211	amod	rich,domain
R3214	T12312	T12313	nsubj	We,determined
R3215	T12214	T12213	punct	-,rich
R3216	T12314	T12313	advmod	next,determined
R3217	T12315	T12316	mark	whether,generate
R3218	T12215	T12208	dobj	stability,decreases
R3219	T12316	T12313	ccomp	generate,determined
R322	T2739	T2740	compound	ES,cells
R3220	T12317	T12318	nmod	p53,ΔPGFP
R3221	T12216	T12208	cc	and,decreases
R3222	T12318	T12321	compound	ΔPGFP,cells
R3223	T12217	T12208	conj	compromises,decreases
R3224	T12319	T12318	punct	+,ΔPGFP
R3225	T12218	T12219	compound	DNA,damage
R3226	T12320	T12318	punct	/,ΔPGFP
R3227	T12321	T12316	nsubj	cells,generate
R3228	T12219	T12221	compound	damage,responses
R3229	T12322	T12321	compound	ES,cells
R323	T2740	T2738	pobj	cells,in
R3230	T12220	T12219	punct	-,damage
R3231	T12323	T12316	aux	could,generate
R3232	T12324	T12325	amod	chimeric,mice
R3233	T12325	T12316	dobj	mice,generate
R3234	T12326	T12316	cc	and,generate
R3235	T12221	T12217	dobj	responses,compromises
R3236	T12327	T12316	conj	transmit,generate
R3237	T12328	T12329	det	the,allele
R3238	T12329	T12327	dobj	allele,transmit
R3239	T12330	T12329	amod	modified,allele
R324	T2741	T2742	aux	to,generate
R3240	T12331	T12327	prep	through,transmit
R3241	T12332	T12333	det	the,germline
R3242	T12222	T12223	advmod	in,vivo
R3243	T12333	T12331	pobj	germline,through
R3244	T12334	T12313	punct	.,determined
R3245	T12223	T12217	advmod	vivo,compromises
R3246	T12336	T12337	nummod	Two,clones
R3247	T12337	T12344	nsubjpass	clones,injected
R3248	T12338	T12339	nmod	p53,ΔPGFP
R3249	T12224	T12225	punct	(,5
R325	T2655	T2656	det	this,approach
R3250	T12339	T12337	compound	ΔPGFP,clones
R3251	T12340	T12339	punct	+,ΔPGFP
R3252	T12341	T12339	punct	/,ΔPGFP
R3253	T12225	T12217	parataxis	5,compromises
R3254	T12342	T12343	compound	ES,cell
R3255	T12343	T12337	compound	cell,clones
R3256	T12226	T12225	punct	),5
R3257	T12345	T12344	auxpass	were,injected
R3258	T12346	T12344	prep	into,injected
R3259	T12227	T12204	punct	.,used
R326	T2742	T2735	advcl	generate,using
R3260	T12347	T12346	pobj	blastocysts,into
R3261	T12348	T12344	cc	and,injected
R3262	T12349	T12350	advmod	highly,chimeric
R3263	T12229	T12230	prep	According,prevent
R3264	T12350	T12351	amod	chimeric,%
R3265	T12351	T12355	amod	%,mice
R3266	T12352	T12351	punct	(,%
R3267	T12353	T12351	punct	>,%
R3268	T12231	T12229	prep	to,According
R3269	T12354	T12351	nummod	80,%
R327	T2743	T2744	amod	mutant,mice
R3270	T12355	T12357	nsubjpass	mice,obtained
R3271	T12356	T12351	punct	),%
R3272	T12357	T12344	conj	obtained,injected
R3273	T12232	T12233	poss	our,hypothesis
R3274	T12358	T12357	auxpass	were,obtained
R3275	T12359	T12357	punct	.,obtained
R3276	T12233	T12231	pobj	hypothesis,to
R3277	T12361	T12362	compound	Heterozygote,pups
R3278	T12362	T12363	nsubjpass	pups,recovered
R3279	T12234	T12230	punct	", ",prevent
R328	T2744	T2742	dobj	mice,generate
R3280	T12364	T12363	auxpass	were,recovered
R3281	T12365	T12363	prep	from,recovered
R3282	T12235	T12236	det	this,mutant
R3283	T12366	T12367	det	the,mating
R3284	T12367	T12365	pobj	mating,from
R3285	T12236	T12230	nsubj	mutant,prevent
R3286	T12237	T12236	amod	hypomorphic,mutant
R3287	T12368	T12369	det	the,chimeras
R3288	T12369	T12367	nmod	chimeras,mating
R3289	T12370	T12367	prep	with,mating
R329	T2745	T2746	det	an,method
R3290	T12371	T12372	compound	WT,mice
R3291	T12372	T12370	pobj	mice,with
R3292	T12373	T12374	punct	(,3C
R3293	T12374	T12363	parataxis	3C,recovered
R3294	T12238	T12230	aux	should,prevent
R3295	T12375	T12374	compound	Figure,3C
R3296	T12376	T12374	punct	),3C
R3297	T12377	T12363	punct	.,recovered
R3298	T12239	T12230	neg	not,prevent
R3299	T12240	T12241	amod	embryonic,development
R33	T474	T473	dobj	mutations,targeting
R330	T2656	T2654	pobj	approach,of
R3300	T12241	T12230	dobj	development,prevent
R3301	T12242	T12230	punct	.,prevent
R3302	T12379	T12380	det	These,data
R3303	T12380	T12381	nsubj	data,demonstrate
R3304	T12244	T12245	nsubj	RMCE,was
R3305	T12382	T12383	mark	that,is
R3306	T12383	T12381	ccomp	is,demonstrate
R3307	T12384	T12385	npadvmod	marker,free
R3308	T12246	T12244	prep	with,RMCE
R3309	T12385	T12387	amod	free,RMCE
R331	T2746	T2733	attr	method,is
R3310	T12386	T12385	punct	-,free
R3311	T12387	T12383	nsubj	RMCE,is
R3312	T12388	T12389	advmod	very,efficient
R3313	T12247	T12248	det	a,plasmid
R3314	T12389	T12383	acomp	efficient,is
R3315	T12390	T12383	prep	in,is
R3316	T12391	T12392	compound	ES,cells
R3317	T12248	T12246	pobj	plasmid,with
R3318	T12392	T12390	pobj	cells,in
R3319	T12393	T12383	cc	and,is
R332	T2747	T2746	amod	inefficient,method
R3320	T12394	T12383	conj	allows,is
R3321	T12249	T12248	compound	p53ΔPGFP,plasmid
R3322	T12395	T12396	compound	germline,transmission
R3323	T12396	T12394	dobj	transmission,allows
R3324	T12397	T12396	prep	of,transmission
R3325	T12250	T12248	compound	replacement,plasmid
R3326	T12398	T12399	det	a,mutation
R3327	T12399	T12397	pobj	mutation,of
R3328	T12251	T12245	advmod	again,was
R3329	T12400	T12399	amod	targeted,mutation
R333	T2748	T2747	punct	", ",inefficient
R3330	T12401	T12402	punct	(,see
R3331	T12402	T12394	parataxis	see,allows
R3332	T12252	T12253	advmod	very,efficient
R3333	T12403	T12402	dobj	Figure,see
R3334	T12404	T12403	nummod	1,Figure
R3335	T12405	T12403	punct	", ",Figure
R3336	T12253	T12245	acomp	efficient,was
R3337	T12406	T12407	compound	path,A
R3338	T12407	T12403	appos	A,Figure
R3339	T12254	T12245	cc	and,was
R334	T2657	T2658	mark	that,tested
R3340	T12408	T12402	punct	),see
R3341	T12409	T12381	punct	.,demonstrate
R3342	T12411	T12412	nsubj	We,determined
R3343	T12255	T12256	compound	western,blots
R3344	T12413	T12412	advmod	next,determined
R3345	T12414	T12415	mark	whether,used
R3346	T12415	T12412	ccomp	used,determined
R3347	T12256	T12257	nsubj	blots,revealed
R3348	T12416	T12417	det	the,approach
R3349	T12417	T12415	nsubjpass	approach,used
R335	T2749	T2747	conj	slow,inefficient
R3350	T12418	T12417	compound	RMCE,approach
R3351	T12257	T12245	conj	revealed,was
R3352	T12419	T12415	aux	could,used
R3353	T12420	T12415	auxpass	be,used
R3354	T12421	T12422	aux	to,target
R3355	T12258	T12259	det	an,band
R3356	T12422	T12415	advcl	target,used
R3357	T12423	T12422	dobj	mutations,target
R3358	T12424	T12423	prep	at,mutations
R3359	T12259	T12257	dobj	band,revealed
R336	T2750	T2749	cc	and,slow
R3360	T12425	T12426	det	the,allele
R3361	T12426	T12424	pobj	allele,at
R3362	T12427	T12426	compound	p53,allele
R3363	T12260	T12259	amod	additional,band
R3364	T12428	T12423	prep	in,mutations
R3365	T12429	T12430	amod	somatic,cells
R3366	T12430	T12428	pobj	cells,in
R3367	T12261	T12259	prep	of,band
R3368	T12431	T12432	punct	(,Figure
R3369	T12432	T12415	parataxis	Figure,used
R337	T2658	T2653	ccomp	tested,is
R3370	T12433	T12432	nummod	1,Figure
R3371	T12262	T12263	det	the,weight
R3372	T12434	T12432	punct	", ",Figure
R3373	T12435	T12436	compound	path,B
R3374	T12436	T12432	appos	B,Figure
R3375	T12263	T12261	pobj	weight,of
R3376	T12437	T12432	punct	),Figure
R3377	T12438	T12412	punct	.,determined
R3378	T12264	T12263	amod	predicted,weight
R3379	T12440	T12441	nsubj	We,verified
R338	T2751	T2749	conj	expensive,slow
R3380	T12265	T12263	amod	molecular,weight
R3381	T12442	T12441	advmod	first,verified
R3382	T12443	T12444	mark	that,transmitted
R3383	T12266	T12267	advmod	only,in
R3384	T12267	T12257	prep	in,revealed
R3385	T12444	T12441	ccomp	transmitted,verified
R3386	T12445	T12446	det	the,locus
R3387	T12268	T12269	nmod	p53,ΔPGFP
R3388	T12446	T12444	nsubjpass	locus,transmitted
R3389	T12447	T12448	npadvmod	RMCE,ready
R339	T2752	T2753	mark	because,occurs
R3390	T12269	T12272	compound	ΔPGFP,cells
R3391	T12448	T12446	amod	ready,locus
R3392	T12449	T12448	punct	-,ready
R3393	T12450	T12446	compound	p53,locus
R3394	T12451	T12446	punct	(,locus
R3395	T12452	T12446	appos	p53RMCE,locus
R3396	T12453	T12444	punct	),transmitted
R3397	T12270	T12269	punct	+,ΔPGFP
R3398	T12454	T12444	aux	could,transmitted
R3399	T12455	T12444	auxpass	be,transmitted
R34	T475	T473	prep	at,targeting
R340	T2753	T2733	advcl	occurs,is
R3400	T12456	T12444	prep	through,transmitted
R3401	T12271	T12269	punct	/,ΔPGFP
R3402	T12457	T12458	det	the,germline
R3403	T12458	T12456	pobj	germline,through
R3404	T12459	T12444	prep	by,transmitted
R3405	T12272	T12267	pobj	cells,in
R3406	T12460	T12459	pcomp	mating,by
R3407	T12461	T12462	nmod	p53RMCE,chimeras
R3408	T12483	T12481	nsubj	this,allowed
R3409	T12462	T12460	dobj	chimeras,mating
R341	T2659	T2658	nsubj	mutations,tested
R3410	T12463	T12461	punct	/,p53RMCE
R3411	T12464	T12461	punct	+,p53RMCE
R3412	T12465	T12460	prep	with,mating
R3413	T12466	T12467	nmod	p53,mice
R3414	T12484	T12485	nsubj	us,generate
R3415	T12467	T12465	pobj	mice,with
R3416	T12468	T12466	punct	+,p53
R3417	T12469	T12466	punct	/,p53
R3418	T12485	T12481	ccomp	generate,allowed
R3419	T12470	T12466	punct	−,p53
R342	T2754	T2755	punct	(,i
R3420	T12471	T12472	punct	(,20
R3421	T12472	T12444	parataxis	20,transmitted
R3422	T12486	T12485	aux	to,generate
R3423	T12473	T12472	punct	),20
R3424	T12474	T12475	punct	(,Figure
R3425	T12475	T12441	parataxis	Figure,verified
R3426	T12487	T12488	nmod	p53RMCE,MEFs
R3427	T12476	T12475	nummod	4,Figure
R3428	T12477	T12475	punct	),Figure
R3429	T12478	T12441	punct	.,verified
R343	T2755	T2753	meta	i,occurs
R3430	T12488	T12485	dobj	MEFs,generate
R3431	T12480	T12481	advmod	Importantly,allowed
R3432	T12489	T12487	punct	/,p53RMCE
R3433	T12482	T12481	punct	", ",allowed
R3434	T12490	T12487	punct	−,p53RMCE
R3435	T12491	T12488	punct	", ",MEFs
R3436	T12492	T12493	dep	which,used
R3437	T12493	T12488	relcl	used,MEFs
R3438	T12589	T12588	pobj	MEFs,in
R3439	T12590	T12578	auxpass	is,activated
R344	T2756	T2755	punct	),i
R3440	T12494	T12493	auxpass	were,used
R3441	T12591	T12578	neg	not,activated
R3442	T12592	T12578	advmod	readily,activated
R3443	T12593	T12578	prep	in,activated
R3444	T12594	T12595	compound	ES,cells
R3445	T12595	T12593	pobj	cells,in
R3446	T12596	T12597	punct	(,18
R3447	T12495	T12496	aux	to,test
R3448	T12597	T12578	parataxis	18,activated
R3449	T12598	T12597	punct	),18
R345	T2757	T2758	amod	homologous,recombination
R3450	T12599	T12578	punct	.,activated
R3451	T12496	T12493	advcl	test,used
R3452	T12601	T12602	det	The,results
R3453	T12497	T12496	dobj	RMCE,test
R3454	T12602	T12603	nsubj	results,suggest
R3455	T12604	T12602	advmod	above,results
R3456	T12498	T12496	prep	at,test
R3457	T12605	T12606	mark	that,be
R3458	T12606	T12603	ccomp	be,suggest
R3459	T12607	T12608	amod	high,levels
R346	T2660	T2658	aux	are,tested
R3460	T12499	T12500	det	the,locus
R3461	T12608	T12606	nsubj	levels,be
R3462	T12609	T12608	prep	of,levels
R3463	T12500	T12498	pobj	locus,at
R3464	T12610	T12609	pobj	p53GFP,of
R3465	T12611	T12606	aux	could,be
R3466	T12612	T12606	dep	tolerated,be
R3467	T12501	T12500	compound	p53,locus
R3468	T12613	T12612	agent	by,tolerated
R3469	T12614	T12615	compound	ES,cells
R347	T2758	T2753	nsubj	recombination,occurs
R3470	T12615	T12613	pobj	cells,by
R3471	T12616	T12612	cc	but,tolerated
R3472	T12502	T12496	prep	in,test
R3473	T12617	T12612	conj	toxic,tolerated
R3474	T12618	T12617	prep	to,toxic
R3475	T12503	T12504	amod	somatic,cells
R3476	T12619	T12618	pobj	MEFs,to
R3477	T12620	T12606	punct	", ",be
R3478	T12621	T12622	mark	so,failed
R3479	T12504	T12502	pobj	cells,in
R348	T2759	T2753	advmod	typically,occurs
R3480	T12622	T12606	advcl	failed,be
R3481	T12505	T12481	punct	.,allowed
R3482	T12623	T12622	mark	that,failed
R3483	T12624	T12622	nsubj	MEFs,failed
R3484	T12507	T12508	nsubj	We,attempted
R3485	T12625	T12626	prep	in,occurred
R3486	T12626	T12624	advcl	occurred,MEFs
R3487	T12627	T12625	pobj	which,in
R3488	T12628	T12629	det	an,RMCE
R3489	T12509	T12508	advmod	first,attempted
R349	T2760	T2753	prep	at,occurs
R3490	T12629	T12626	nsubj	RMCE,occurred
R3491	T12630	T12626	aux	had,occurred
R3492	T12631	T12632	aux	to,proliferate
R3493	T12632	T12622	xcomp	proliferate,failed
R3494	T12510	T12508	dobj	RMCE,attempted
R3495	T12633	T12603	punct	.,suggest
R3496	T12635	T12636	aux	To,test
R3497	T12636	T12637	advcl	test,electroporated
R3498	T12511	T12508	prep	in,attempted
R3499	T12638	T12639	det	this,possibility
R35	T476	T477	det	the,locus
R350	T2661	T2658	prep	in,tested
R3500	T12639	T12636	dobj	possibility,test
R3501	T12640	T12637	punct	", ",electroporated
R3502	T12512	T12511	pobj	MEFs,in
R3503	T12641	T12642	nmod	p53RMCE,MEFs
R3504	T12642	T12637	nsubjpass	MEFs,electroporated
R3505	T12643	T12641	punct	/,p53RMCE
R3506	T12513	T12508	prep	by,attempted
R3507	T12644	T12641	punct	−,p53RMCE
R3508	T12645	T12637	auxpass	were,electroporated
R3509	T12646	T12637	prep	with,electroporated
R351	T2761	T2762	amod	low,frequency
R3510	T12514	T12513	pcomp	electroporating,by
R3511	T12647	T12648	det	the,construct
R3512	T12648	T12646	pobj	construct,with
R3513	T12649	T12648	compound	p53GFP,construct
R3514	T12515	T12516	nmod	p53RMCE,MEFs
R3515	T12650	T12648	compound	replacement,construct
R3516	T12651	T12637	prep	with,electroporated
R3517	T12516	T12514	dobj	MEFs,electroporating
R3518	T12652	T12651	cc	or,with
R3519	T12653	T12651	conj	without,with
R352	T2762	T2760	pobj	frequency,at
R3520	T12654	T12655	det	a,plasmid
R3521	T12517	T12515	punct	/,p53RMCE
R3522	T12655	T12653	pobj	plasmid,without
R3523	T12656	T12657	compound	Cre,expression
R3524	T12657	T12655	compound	expression,plasmid
R3525	T12518	T12515	punct	−,p53RMCE
R3526	T12658	T12657	punct	-,expression
R3527	T12659	T12637	punct	", ",electroporated
R3528	T12660	T12661	advmod	then,analyzed
R3529	T12661	T12637	dep	analyzed,electroporated
R353	T2763	T2753	prep	in,occurs
R3530	T12519	T12514	prep	with,electroporating
R3531	T12662	T12661	prep	by,analyzed
R3532	T12663	T12664	compound	fluorescence,microscopy
R3533	T12520	T12521	det	a,plasmid
R3534	T12664	T12662	pobj	microscopy,by
R3535	T12665	T12666	nummod	48,h
R3536	T12666	T12667	npadvmod	h,after
R3537	T12521	T12519	pobj	plasmid,with
R3538	T12667	T12661	prep	after,analyzed
R3539	T12668	T12667	pobj	electroporation,after
R354	T2764	T2765	compound	ES,cells
R3540	T12669	T12637	punct	.,electroporated
R3541	T12522	T12523	compound	Cre,expression
R3542	T12671	T12672	det	The,experiment
R3543	T12672	T12673	nsubjpass	experiment,done
R3544	T12523	T12521	compound	expression,plasmid
R3545	T12674	T12673	auxpass	was,done
R3546	T12675	T12673	prep	without,done
R3547	T12524	T12523	punct	-,expression
R3548	T12676	T12675	pobj	selection,without
R3549	T12677	T12678	aux	to,enable
R355	T2765	T2763	pobj	cells,in
R3550	T12678	T12673	advcl	enable,done
R3551	T12679	T12678	dobj	observation,enable
R3552	T12680	T12679	prep	of,observation
R3553	T12525	T12521	cc	and,plasmid
R3554	T12681	T12680	pobj	cells,of
R3555	T12682	T12678	prep	under,enable
R3556	T12526	T12527	det	the,plasmid
R3557	T12683	T12682	pobj	conditions,under
R3558	T12684	T12685	advmod	where,derive
R3559	T12685	T12683	relcl	derive,conditions
R356	T2766	T2753	punct	", ",occurs
R3560	T12686	T12687	det	a,failure
R3561	T12527	T12521	conj	plasmid,plasmid
R3562	T12687	T12685	nsubj	failure,derive
R3563	T12688	T12689	aux	to,proliferate
R3564	T12528	T12527	compound	p53GFP,plasmid
R3565	T12689	T12687	acl	proliferate,failure
R3566	T12690	T12685	aux	would,derive
R3567	T12691	T12685	neg	not,derive
R3568	T12529	T12521	punct	", ",plasmid
R3569	T12692	T12685	prep	from,derive
R357	T2662	T2663	det	a,setting
R3570	T12693	T12694	nmod	FIAU,toxicity
R3571	T12694	T12692	pobj	toxicity,from
R3572	T12530	T12521	acl	followed,plasmid
R3573	T12531	T12530	prep	by,followed
R3574	T12532	T12531	pobj	selection,by
R3575	T12695	T12693	cc	or,FIAU
R3576	T12533	T12532	prep	with,selection
R3577	T12696	T12693	conj	ganciclovir,FIAU
R3578	T12697	T12692	cc	but,from
R3579	T12698	T12697	advmod	rather,but
R358	T2767	T2753	advcl	requiring,occurs
R3580	T12534	T12533	pobj	FIAU,with
R3581	T12699	T12700	advmod	solely,from
R3582	T12700	T12692	conj	from,from
R3583	T12701	T12702	det	the,effects
R3584	T12535	T12534	cc	or,FIAU
R3585	T12702	T12700	pobj	effects,from
R3586	T12703	T12702	prep	of,effects
R3587	T12536	T12534	conj	ganciclovir,FIAU
R3588	T12704	T12703	pobj	p53GFP,of
R3589	T12705	T12673	punct	.,done
R359	T2768	T2769	amod	sophisticated,schemes
R3590	T12537	T12508	punct	.,attempted
R3591	T12707	T12708	nsubj	We,observed
R3592	T12539	T12540	advmod	Strikingly,identified
R3593	T12709	T12710	det	a,cells
R3594	T12710	T12708	dobj	cells,observed
R3595	T12541	T12540	punct	", ",identified
R3596	T12711	T12710	amod	few,cells
R3597	T12542	T12543	det	no,clones
R3598	T12712	T12710	amod	fluorescent,cells
R3599	T12713	T12714	advmod	only,when
R36	T477	T475	pobj	locus,at
R360	T2663	T2661	pobj	setting,in
R3600	T12714	T12715	advmod	when,co-electroporated
R3601	T12715	T12710	advcl	co-electroporated,cells
R3602	T12543	T12540	nsubjpass	clones,identified
R3603	T12716	T12717	det	the,plasmid
R3604	T12717	T12715	nsubjpass	plasmid,co-electroporated
R3605	T12718	T12719	compound	Cre,expression
R3606	T12544	T12543	prep	with,clones
R3607	T12719	T12717	compound	expression,plasmid
R3608	T12720	T12715	auxpass	was,co-electroporated
R3609	T12721	T12715	punct	", ",co-electroporated
R361	T2769	T2767	dobj	schemes,requiring
R3610	T12545	T12546	det	an,allele
R3611	T12722	T12715	advcl	suggesting,co-electroporated
R3612	T12723	T12724	mark	that,resulted
R3613	T12724	T12722	ccomp	resulted,suggesting
R3614	T12546	T12544	pobj	allele,with
R3615	T12725	T12726	amod	such,cells
R3616	T12726	T12724	nsubj	cells,resulted
R3617	T12727	T12724	prep	from,resulted
R3618	T12547	T12546	amod	exchanged,allele
R3619	T12728	T12727	pobj	RMCE,from
R362	T2770	T2769	compound	selection,schemes
R3620	T12729	T12708	punct	.,observed
R3621	T12548	T12540	auxpass	were,identified
R3622	T12731	T12732	advmod	Importantly,had
R3623	T12733	T12732	punct	", ",had
R3624	T12549	T12550	punct	(,shown
R3625	T12734	T12735	det	the,cells
R3626	T12735	T12732	nsubj	cells,had
R3627	T12736	T12735	amod	rare,cells
R3628	T12550	T12540	parataxis	shown,identified
R3629	T12737	T12735	amod	fluorescent,cells
R363	T2771	T2769	cc	and,schemes
R3630	T12738	T12739	det	a,appearance
R3631	T12739	T12732	dobj	appearance,had
R3632	T12551	T12550	nsubj	data,shown
R3633	T12740	T12739	amod	flat,appearance
R3634	T12741	T12739	punct	", ",appearance
R3635	T12742	T12739	punct	‘,appearance
R3636	T12552	T12550	neg	not,shown
R3637	T12743	T12744	amod	fried,egg
R3638	T12744	T12739	nmod	egg,appearance
R3639	T12745	T12744	punct	-,egg
R364	T2772	T2769	conj	screening,schemes
R3640	T12553	T12550	punct	),shown
R3641	T12746	T12739	punct	’,appearance
R3642	T12747	T12739	amod	typical,appearance
R3643	T12748	T12747	prep	of,typical
R3644	T12554	T12540	punct	.,identified
R3645	T12749	T12750	amod	senescent,cells
R3646	T12750	T12748	pobj	cells,of
R3647	T12751	T12752	punct	(,5A
R3648	T12556	T12557	nsubj	RMCE,showed
R3649	T12752	T12732	parataxis	5A,had
R365	T2664	T2663	amod	genomic,setting
R3650	T12753	T12752	compound	Figure,5A
R3651	T12754	T12752	punct	),5A
R3652	T12755	T12732	punct	", ",had
R3653	T12756	T12732	cc	and,had
R3654	T12757	T12758	advmod	when,observed
R3655	T12758	T12761	advcl	observed,detached
R3656	T12558	T12556	prep	with,RMCE
R3657	T12759	T12758	nsubjpass	plates,observed
R3658	T12760	T12758	auxpass	were,observed
R3659	T12761	T12732	conj	detached,had
R366	T2773	T2772	prep	of,screening
R3660	T12762	T12763	nummod	5,days
R3661	T12559	T12558	pobj	p53GFP,with
R3662	T12763	T12764	npadvmod	days,later
R3663	T12764	T12758	advmod	later,observed
R3664	T12765	T12761	punct	", ",detached
R3665	T12560	T12556	prep	in,RMCE
R3666	T12766	T12767	det	the,cells
R3667	T12767	T12761	nsubj	cells,detached
R3668	T12561	T12562	compound	ES,cells
R3669	T12768	T12761	aux	had,detached
R367	T2774	T2773	pobj	hundreds,of
R3670	T12769	T12761	punct	.,detached
R3671	T12562	T12560	pobj	cells,in
R3672	T12771	T12772	advmod	Altogether,suggest
R3673	T12773	T12772	punct	", ",suggest
R3674	T12563	T12564	mark	that,expressed
R3675	T12774	T12775	det	the,results
R3676	T12775	T12772	nsubj	results,suggest
R3677	T12776	T12777	mark	that,give
R3678	T12564	T12557	ccomp	expressed,showed
R3679	T12777	T12772	ccomp	give,suggest
R368	T2665	T2658	cc	and,tested
R3680	T12778	T12777	nsubj	RMCE,give
R3681	T12779	T12777	aux	can,give
R3682	T12565	T12564	nsubjpass	p53GFP,expressed
R3683	T12780	T12777	dobj	rise,give
R3684	T12781	T12777	prep	to,give
R3685	T12782	T12783	npadvmod	p53GFP,expressing
R3686	T12566	T12564	auxpass	is,expressed
R3687	T12783	T12785	amod	expressing,MEFs
R3688	T12784	T12783	punct	-,expressing
R3689	T12567	T12564	prep	at,expressed
R369	T2775	T2774	prep	of,hundreds
R3690	T12785	T12781	pobj	MEFs,to
R3691	T12786	T12777	punct	", ",give
R3692	T12787	T12777	cc	but,give
R3693	T12788	T12789	nsubj	they,die
R3694	T12568	T12569	amod	high,levels
R3695	T12789	T12777	conj	die,give
R3696	T12790	T12789	advmod	rapidly,die
R3697	T12569	T12567	pobj	levels,at
R3698	T12791	T12789	prep	owing,die
R3699	T12792	T12791	prep	to,owing
R37	T478	T477	compound	p53,locus
R370	T2776	T2775	pobj	clones,of
R3700	T12793	T12794	compound	p53GFP,toxicity
R3701	T12570	T12571	punct	(,3A
R3702	T12794	T12792	pobj	toxicity,to
R3703	T12795	T12772	punct	.,suggest
R3704	T12571	T12564	parataxis	3A,expressed
R3705	T12797	T12798	nsubj	We,performed
R3706	T12799	T12798	advmod	also,performed
R3707	T12572	T12571	compound	Figure,3A
R3708	T12800	T12798	dobj	RMCE,performed
R3709	T12573	T12571	punct	),3A
R371	T2777	T2778	aux	to,identify
R3710	T12574	T12557	punct	", ",showed
R3711	T12801	T12798	prep	in,performed
R3712	T12802	T12803	nmod	p53RMCE,MEFs
R3713	T12575	T12557	cc	and,showed
R3714	T12803	T12801	pobj	MEFs,in
R3715	T12804	T12802	punct	/,p53RMCE
R3716	T12576	T12577	mark	as,mentioned
R3717	T12805	T12802	punct	−,p53RMCE
R3718	T12806	T12798	prep	with,performed
R3719	T12577	T12578	advcl	mentioned,activated
R372	T2666	T2658	conj	expressed,tested
R3720	T12807	T12808	det	the,construct
R3721	T12808	T12806	pobj	construct,with
R3722	T12578	T12557	conj	activated,showed
R3723	T12809	T12808	compound	p53ΔPGFP,construct
R3724	T12810	T12798	punct	.,performed
R3725	T12579	T12577	advmod	before,mentioned
R3726	T12812	T12813	npadvmod	p53ΔPGFP,expressing
R3727	T12813	T12815	amod	expressing,MEFs
R3728	T12814	T12813	punct	-,expressing
R3729	T12580	T12578	punct	", ",activated
R373	T2778	T2767	advcl	identify,requiring
R3730	T12815	T12816	nsubj	MEFs,were
R3731	T12581	T12582	det	the,pathway
R3732	T12817	T12816	acomp	viable,were
R3733	T12818	T12816	punct	", ",were
R3734	T12819	T12816	conj	recovered,were
R3735	T12582	T12578	nsubjpass	pathway,activated
R3736	T12820	T12819	prep	with,recovered
R3737	T12821	T12822	det	an,efficiency
R3738	T12583	T12582	compound	p53,pathway
R3739	T12822	T12820	pobj	efficiency,with
R374	T2779	T2780	det	the,mutant
R3740	T12823	T12822	prep	of,efficiency
R3741	T12824	T12825	punct	∼,40
R3742	T12584	T12585	dep	that,activated
R3743	T12825	T12826	nummod	40,%
R3744	T12826	T12823	pobj	%,of
R3745	T12827	T12819	punct	", ",recovered
R3746	T12585	T12582	relcl	activated,pathway
R3747	T12828	T12819	cc	and,recovered
R3748	T12829	T12819	punct	", ",recovered
R3749	T12830	T12831	mark	as,expected
R375	T2780	T2778	dobj	mutant,identify
R3750	T12586	T12585	aux	can,activated
R3751	T12831	T12832	advcl	expected,expressed
R3752	T12832	T12819	conj	expressed,recovered
R3753	T12833	T12831	prep	from,expected
R3754	T12587	T12585	auxpass	be,activated
R3755	T12834	T12835	compound	ES,cell
R3756	T12835	T12836	compound	cell,experiments
R3757	T12836	T12833	pobj	experiments,from
R3758	T12837	T12832	punct	", ",expressed
R3759	T12838	T12839	det	a,protein
R376	T2667	T2666	prep	from,expressed
R3760	T12588	T12585	prep	in,activated
R3761	T12839	T12832	dobj	protein,expressed
R3762	T12840	T12839	compound	p53ΔPGFP,protein
R3763	T12841	T12832	prep	at,expressed
R3764	T12842	T12843	advmod	much,lower
R3765	T12906	T12878	aux	did,arrest
R3766	T12843	T12844	amod	lower,levels
R3767	T12844	T12841	pobj	levels,at
R3768	T12845	T12844	prep	than,levels
R3769	T12846	T12845	pobj	p53WT,than
R377	T2781	T2780	amod	desired,mutant
R3770	T12907	T12878	neg	not,arrest
R3771	T12847	T12848	punct	(,5B
R3772	T12848	T12832	parataxis	5B,expressed
R3773	T12849	T12848	compound	Figure,5B
R3774	T12850	T12848	punct	),5B
R3775	T12908	T12909	nmod	p53ΔPGFP,MEFs
R3776	T12851	T12816	punct	.,were
R3777	T12909	T12878	dobj	MEFs,arrest
R3778	T12853	T12854	prep	Unlike,are
R3779	T12855	T12856	compound	WT,MEFs
R378	T2782	T2753	punct	;,occurs
R3780	T12910	T12908	punct	/,p53ΔPGFP
R3781	T12856	T12853	pobj	MEFs,Unlike
R3782	T12857	T12854	punct	", ",are
R3783	T12858	T12859	compound	p53ΔP,ΔP
R3784	T12911	T12908	punct	−,p53ΔPGFP
R3785	T12859	T12861	compound	ΔP,MEFs
R3786	T12860	T12859	punct	/,ΔP
R3787	T12912	T12913	punct	(,5C
R3788	T12861	T12854	nsubj	MEFs,are
R3789	T12862	T12854	acomp	unable,are
R379	T2783	T2784	punct	(,ii
R3790	T12863	T12864	aux	to,arrest
R3791	T12913	T12878	parataxis	5C,arrest
R3792	T12864	T12862	xcomp	arrest,unable
R3793	T12865	T12864	dobj	cycling,arrest
R3794	T12866	T12864	prep	after,arrest
R3795	T12914	T12913	compound	Figure,5C
R3796	T12867	T12866	pobj	irradiation,after
R3797	T12868	T12869	punct	(,5
R3798	T12869	T12854	parataxis	5,are
R3799	T12915	T12913	punct	),5C
R38	T479	T473	advcl	using,targeting
R380	T2668	T2669	det	the,promoter
R3800	T12870	T12869	punct	),5
R3801	T12871	T12854	punct	.,are
R3802	T12916	T12874	punct	.,found
R3803	T12873	T12874	advmod	Likewise,found
R3804	T12918	T12919	det	These,data
R3805	T12875	T12874	punct	", ",found
R3806	T12876	T12874	nsubj	we,found
R3807	T12877	T12878	mark	that,arrest
R3808	T12919	T12920	nsubj	data,show
R3809	T12878	T12874	ccomp	arrest,found
R381	T2784	T2785	meta	ii,plasmids
R3810	T12921	T12922	mark	that,enables
R3811	T12879	T12880	compound	irradiation,doses
R3812	T12880	T12878	nsubj	doses,arrest
R3813	T12881	T12882	dep	that,arrested
R3814	T12882	T12880	relcl	arrested,doses
R3815	T12922	T12920	ccomp	enables,show
R3816	T12883	T12884	nmod	p53RMCE,MEFs
R3817	T12884	T12882	dobj	MEFs,arrested
R3818	T12923	T12924	det	a,reaction
R3819	T12885	T12883	punct	/,p53RMCE
R382	T2669	T2667	pobj	promoter,from
R3820	T12886	T12883	punct	−,p53RMCE
R3821	T12887	T12880	punct	(,doses
R3822	T12924	T12922	nsubj	reaction,enables
R3823	T12888	T12889	dep	which,express
R3824	T12889	T12880	relcl	express,doses
R3825	T12890	T12891	det	a,p53
R3826	T12925	T12924	amod	single,reaction
R3827	T12891	T12889	dobj	p53,express
R3828	T12892	T12893	amod	wild,type
R3829	T12926	T12927	compound	RMCE,ASAP
R383	T2785	T2794	nsubjpass	plasmids,required
R3830	T12893	T12891	compound	type,p53
R3831	T12894	T12893	punct	-,type
R3832	T12895	T12891	prep	from,p53
R3833	T12927	T12924	compound	ASAP,reaction
R3834	T12896	T12897	det	the,locus
R3835	T12897	T12895	pobj	locus,from
R3836	T12898	T12899	npadvmod	RMCE,ready
R3837	T12928	T12927	punct	-,ASAP
R3838	T12899	T12897	amod	ready,locus
R3839	T12900	T12899	punct	-,ready
R384	T2786	T2784	punct	),ii
R3840	T12901	T12878	punct	", ",arrest
R3841	T12929	T12924	prep	in,reaction
R3842	T12902	T12878	parataxis	see,arrest
R3843	T12903	T12902	dobj	Figure,see
R3844	T12930	T12931	amod	heterozygous,MEFs
R3845	T12904	T12903	nummod	2,Figure
R3846	T12905	T12878	punct	),arrest
R3847	T12931	T12929	pobj	MEFs,in
R3848	T12932	T12922	dobj	detection,enables
R3849	T12933	T12932	prep	of,detection
R385	T2670	T2669	amod	endogenous,promoter
R3850	T13012	T13013	prep	in,accelerate
R3851	T12934	T12935	det	a,phenotype
R3852	T13013	T13001	conj	accelerate,enables
R3853	T13014	T13015	amod	heterozygous,MEFs
R3854	T12935	T12933	pobj	phenotype,of
R3855	T13015	T13012	pobj	MEFs,in
R3856	T13016	T13013	aux	to,accelerate
R3857	T13017	T13018	amod	phenotypic,analyses
R3858	T12936	T12935	amod	recessive,phenotype
R3859	T13018	T13013	dobj	analyses,accelerate
R386	T2787	T2785	amod	large,plasmids
R3860	T13019	T12990	punct	.,report
R3861	T13021	T13022	det	The,success
R3862	T13022	T13023	nsubj	success,relied
R3863	T12937	T12920	punct	.,show
R3864	T13024	T13022	prep	of,success
R3865	T13025	T13026	compound	RMCE,ASAP
R3866	T12939	T12940	det	The,results
R3867	T13026	T13024	pobj	ASAP,of
R3868	T13027	T13026	punct	-,ASAP
R3869	T13028	T13023	prep	on,relied
R387	T2788	T2789	punct	(,kb
R3870	T13029	T13030	det	the,use
R3871	T13030	T13028	pobj	use,on
R3872	T12940	T12941	nsubj	results,confirm
R3873	T13031	T13030	amod	integrated,use
R3874	T13032	T13030	prep	of,use
R3875	T13033	T13034	amod	inverted,sites
R3876	T12942	T12943	mark	that,leads
R3877	T13034	T13032	pobj	sites,of
R3878	T13035	T13036	amod	heterologous,loxP
R3879	T13036	T13034	compound	loxP,sites
R388	T2789	T2785	parataxis	kb,plasmids
R3880	T13037	T13034	punct	", ",sites
R3881	T12943	T12941	ccomp	leads,confirm
R3882	T13038	T13039	det	a,marker
R3883	T13039	T13034	conj	marker,sites
R3884	T12944	T12943	csubj	deleting,leads
R3885	T13040	T13041	amod	positive,negative
R3886	T13041	T13043	nmod	negative,selection
R3887	T13042	T13041	punct	/,negative
R3888	T12945	T12946	det	the,domain
R3889	T13043	T13039	compound	selection,marker
R389	T2790	T2791	quantmod	15,20
R3890	T13044	T13045	dep	that,preserves
R3891	T13045	T13039	relcl	preserves,marker
R3892	T13046	T13047	det	the,capacity
R3893	T12946	T12944	dobj	domain,deleting
R3894	T13047	T13045	dobj	capacity,preserves
R3895	T13048	T13047	compound	germline,capacity
R3896	T13049	T13047	prep	of,capacity
R3897	T12947	T12948	npadvmod	proline,rich
R3898	T13050	T13051	compound	ES,cells
R3899	T13051	T13049	pobj	cells,of
R39	T480	T481	amod	homologous,recombination
R390	T2671	T2658	punct	", ",tested
R3900	T13052	T13039	punct	", ",marker
R3901	T12948	T12946	amod	rich,domain
R3902	T13053	T13039	cc	and,marker
R3903	T13054	T13039	punct	", ",marker
R3904	T12949	T12943	prep	to,leads
R3905	T12950	T12951	advmod	less,active
R3906	T13055	T13056	prep	for,existence
R3907	T13056	T13039	conj	existence,marker
R3908	T13057	T13058	amod	somatic,cells
R3909	T12951	T12952	amod	active,p53
R391	T2791	T2789	nummod	20,kb
R3910	T13058	T13055	pobj	cells,for
R3911	T13059	T13056	punct	", ",existence
R3912	T13060	T13056	det	the,existence
R3913	T13061	T13056	prep	of,existence
R3914	T13062	T13063	det	a,allele
R3915	T13063	T13061	pobj	allele,of
R3916	T12952	T12949	pobj	p53,to
R3917	T13064	T13063	amod	knock,allele
R3918	T13065	T13064	punct	-,knock
R3919	T13066	T13064	prt	out,knock
R392	T2792	T2791	punct	–,20
R3920	T12953	T12952	prep	with,p53
R3921	T13067	T13063	prep	of,allele
R3922	T13068	T13069	det	the,gene
R3923	T13069	T13067	pobj	gene,of
R3924	T12954	T12955	amod	impaired,control
R3925	T13070	T13069	prep	of,gene
R3926	T13071	T13070	pobj	interest,of
R3927	T12955	T12953	pobj	control,with
R3928	T13072	T13023	punct	.,relied
R3929	T13074	T13075	det	These,characteristics
R393	T2793	T2789	punct	),kb
R3930	T12956	T12957	compound	cell,cycle
R3931	T13075	T13076	nsubj	characteristics,make
R3932	T13077	T13076	aux	should,make
R3933	T12957	T12955	compound	cycle,control
R3934	T13078	T13079	compound	RMCE,ASAP
R3935	T13079	T13081	nsubj	ASAP,technology
R3936	T13080	T13079	punct	-,ASAP
R3937	T12958	T12941	punct	", ",confirm
R3938	T13081	T13076	ccomp	technology,make
R3939	T13082	T13081	det	a,technology
R394	T2672	T2658	advcl	ensuring,tested
R3940	T13083	T13081	amod	robust,technology
R3941	T12959	T12941	cc	and,confirm
R3942	T13084	T13083	cc	and,robust
R3943	T13085	T13083	conj	general,robust
R3944	T13086	T13081	prep	for,technology
R3945	T12960	T12961	advmod	also,indicate
R3946	T13087	T13086	pobj	analysis,for
R3947	T13088	T13087	prep	of,analysis
R3948	T13089	T13090	amod	mammalian,genes
R3949	T12961	T12941	conj	indicate,confirm
R395	T2794	T2753	conj	required,occurs
R3950	T13090	T13088	pobj	genes,of
R3951	T13091	T13087	prep	under,analysis
R3952	T12962	T12963	mark	that,alter
R3953	T13092	T13091	pobj	conditions,under
R3954	T13093	T13094	dep	that,preserve
R3955	T13094	T13092	relcl	preserve,conditions
R3956	T12963	T12961	ccomp	alter,indicate
R3957	T13095	T13096	amod	normal,mechanisms
R3958	T13096	T13094	dobj	mechanisms,preserve
R3959	T13097	T13096	compound	control,mechanisms
R396	T2795	T2785	punct	", ",plasmids
R3960	T13098	T13094	prep	in,preserve
R3961	T12964	T12965	det	a,fusion
R3962	T13099	T13100	amod	different,tissues
R3963	T13100	T13098	pobj	tissues,in
R3964	T12965	T12963	nsubj	fusion,alter
R3965	T13101	T13076	punct	.,make
R3966	T12966	T12965	nmod	GFP,fusion
R3967	T13103	T13104	prep	In,used
R3968	T12967	T12965	nmod	C,fusion
R3969	T13105	T13103	pobj	addition,In
R397	T2796	T2797	advmod	often,difficult
R3970	T13106	T13104	punct	", ",used
R3971	T13107	T13108	compound	RMCE,ASAP
R3972	T12968	T12967	punct	-,C
R3973	T13108	T13104	nsubjpass	ASAP,used
R3974	T13109	T13108	punct	-,ASAP
R3975	T12969	T12967	amod	terminal,C
R3976	T13110	T13104	aux	could,used
R3977	T13111	T13104	auxpass	be,used
R3978	T13112	T13113	aux	to,generate
R3979	T12970	T12963	aux	can,alter
R398	T2673	T2674	amod	physiological,levels
R3980	T13113	T13104	advcl	generate,used
R3981	T13114	T13115	amod	fibroblastic,lines
R3982	T13115	T13113	dobj	lines,generate
R3983	T12971	T12963	advmod	dramatically,alter
R3984	T13116	T13115	compound	cell,lines
R3985	T13117	T13115	acl	tailored,lines
R3986	T12972	T12973	compound	p53,regulation
R3987	T12973	T12963	dobj	regulation,alter
R3988	T13118	T13117	prep	for,tailored
R3989	T12974	T12941	punct	.,confirm
R399	T2797	T2785	amod	difficult,plasmids
R3990	T13119	T13120	det	the,targeting
R3991	T13120	T13118	pobj	targeting,for
R3992	T13121	T13120	amod	repeated,targeting
R3993	T12976	T12977	det	A,summary
R3994	T13122	T13120	prep	of,targeting
R3995	T13123	T13124	advmod	widely,studied
R3996	T13124	T13125	amod	studied,genes
R3997	T12977	T12978	nsubjpass	summary,presented
R3998	T13125	T13122	pobj	genes,of
R3999	T13126	T13125	punct	(,genes
R40	T481	T479	dobj	recombination,using
R400	T2798	T2799	aux	to,clone
R4000	T13127	T13125	appos	p53,genes
R4001	T12979	T12977	prep	of,summary
R4002	T13128	T13127	punct	", ",p53
R4003	T13129	T13130	compound	c,myc
R4004	T13130	T13127	conj	myc,p53
R4005	T12980	T12981	poss	our,results
R4006	T13131	T13130	punct	-,myc
R4007	T13132	T13130	punct	", ",myc
R4008	T13133	T13134	compound	NF,KB
R4009	T12981	T12979	pobj	results,of
R401	T2674	T2672	dobj	levels,ensuring
R4010	T13134	T13130	conj	KB,myc
R4011	T13135	T13134	punct	-,KB
R4012	T13136	T13134	punct	", ",KB
R4013	T12982	T12978	auxpass	is,presented
R4014	T13137	T13134	conj	etc.,KB
R4015	T13138	T13104	punct	),used
R4016	T13139	T13104	punct	.,used
R4017	T12983	T12978	prep	in,presented
R4018	T12984	T12983	pobj	Table,in
R4019	T12985	T12984	nummod	1,Table
R402	T2799	T2797	advcl	clone,difficult
R4020	T12986	T12978	punct	.,presented
R4021	T12988	T12989	det	These,data
R4022	T12989	T12990	nsubj	data,report
R4023	T12991	T12992	det	the,development
R4024	T12992	T12990	dobj	development,report
R4025	T12993	T12992	cc	and,development
R4026	T12994	T12992	conj	implementation,development
R4027	T12995	T12992	prep	of,development
R4028	T12996	T12997	det	an,approach
R4029	T12997	T12995	pobj	approach,of
R403	T2800	T2794	punct	", ",required
R4030	T12998	T12999	amod	improved,RMCE
R4031	T12999	T12997	compound	RMCE,approach
R4032	T13000	T13001	dep	that,enables
R4033	T13001	T12997	relcl	enables,approach
R4034	T13002	T13003	amod	efficient,modification
R4035	T13003	T13001	dobj	modification,enables
R4036	T13004	T13003	compound	allele,modification
R4037	T13005	T13001	prep	in,enables
R4038	T13006	T13007	compound	ES,cells
R4039	T13007	T13005	pobj	cells,in
R404	T2675	T2674	compound	expression,levels
R4040	T13008	T13009	aux	to,generate
R4041	T13009	T13001	advcl	generate,enables
R4042	T13010	T13009	dobj	mice,generate
R4043	T13011	T13001	cc	and,enables
R405	T2676	T2674	cc	and,levels
R406	T2801	T2794	auxpass	are,required
R407	T2677	T2678	amod	correct,profiles
R408	T2802	T2803	aux	to,increase
R409	T2803	T2794	advcl	increase,required
R41	T482	T479	prep	in,using
R410	T2678	T2674	conj	profiles,levels
R411	T2804	T2805	amod	targeting,efficiency
R412	T2805	T2803	dobj	efficiency,increase
R413	T2806	T2794	cc	and,required
R414	T2679	T2680	amod	spatio,temporal
R415	T2807	T2808	punct	(,iii
R416	T2808	T2809	meta	iii,generate
R417	T2680	T2678	amod	temporal,profiles
R418	T2809	T2794	conj	generate,required
R419	T2810	T2808	punct	),iii
R42	T483	T484	compound	ES,cells
R420	T2811	T2809	csubj	breeding,generate
R421	T2681	T2680	punct	-,temporal
R422	T2812	T2811	dobj	mice,breeding
R423	T2813	T2811	prep	to,breeding
R424	T2814	T2813	pobj	homozygosity,to
R425	T2682	T2653	punct	.,is
R426	T2815	T2811	cc	and,breeding
R427	T2816	T2811	conj	housing,breeding
R428	T2684	T2685	mark	As,observed
R429	T2817	T2818	det	a,colony
R43	T484	T482	pobj	cells,in
R430	T2818	T2816	dobj	colony,housing
R431	T2819	T2818	compound	mouse,colony
R432	T2820	T2821	amod	further,delays
R433	T2821	T2809	dobj	delays,generate
R434	T2685	T2700	advcl	observed,need
R435	T2822	T2821	cc	and,delays
R436	T2823	T2821	conj	costs,delays
R437	T2824	T2733	punct	.,is
R438	T2686	T2687	amod	significant,differences
R439	T2826	T2827	amod	Such,limitations
R44	T485	T486	aux	to,generate
R440	T2687	T2685	nsubjpass	differences,observed
R441	T2827	T2828	nsubj	limitations,make
R442	T2829	T2830	det	the,targeting
R443	T2688	T2687	prep	between,differences
R444	T2830	T2832	nsubj	targeting,task
R445	T2831	T2830	amod	repeated,targeting
R446	T2832	T2828	ccomp	task,make
R447	T2689	T2688	pobj	phenotypes,between
R448	T2833	T2830	prep	of,targeting
R449	T2834	T2835	det	a,locus
R45	T486	T479	advcl	generate,using
R450	T2835	T2833	pobj	locus,of
R451	T2690	T2689	prep	from,phenotypes
R452	T2836	T2832	det	a,task
R453	T2837	T2838	advmod	technically,daunting
R454	T2838	T2832	amod	daunting,task
R455	T2691	T2692	amod	targeted,mutants
R456	T2839	T2838	cc	and,daunting
R457	T2840	T2841	advmod	economically,impractical
R458	T2841	T2838	conj	impractical,daunting
R459	T2842	T2828	punct	.,make
R46	T487	T488	amod	mutant,mice
R460	T2844	T2845	nsubjpass	Improvements,needed
R461	T2692	T2690	pobj	mutants,from
R462	T2846	T2844	prep	in,Improvements
R463	T2693	T2692	compound	p53,mutants
R464	T2847	T2848	amod	current,technologies
R465	T2848	T2846	pobj	technologies,in
R466	T2849	T2845	auxpass	are,needed
R467	T2694	T2695	advmod	in,vivo
R468	T2850	T2851	aux	to,enable
R469	T2851	T2845	advcl	enable,needed
R47	T488	T486	dobj	mice,generate
R470	T2907	T2905	compound	read,out
R471	T2852	T2853	amod	such,analyses
R472	T2853	T2851	dobj	analyses,enable
R473	T2854	T2855	aux	to,applied
R474	T2855	T2851	xcomp	applied,enable
R475	T2908	T2905	punct	-,out
R476	T2856	T2855	auxpass	be,applied
R477	T2857	T2855	dative	to,applied
R478	T2858	T2859	det	the,p53
R479	T2909	T2905	prep	than,out
R48	T489	T461	punct	.,gained
R480	T2859	T2857	pobj	p53,to
R481	T2860	T2859	cc	or,p53
R482	T2910	T2911	amod	equivalent,outs
R483	T2861	T2862	amod	other,genes
R484	T2862	T2859	conj	genes,p53
R485	T2863	T2845	punct	.,needed
R486	T2911	T2909	pobj	outs,than
R487	T2865	T2866	csubj	Developing,is
R488	T2912	T2911	compound	gene,outs
R489	T2867	T2865	dobj	methods,Developing
R49	T491	T492	mark	Although,informative
R490	T2868	T2869	aux	to,increase
R491	T2913	T2911	compound	knock,outs
R492	T2869	T2867	advcl	increase,methods
R493	T2870	T2871	amod	targeting,efficiency
R494	T2871	T2869	dobj	efficiency,increase
R495	T2872	T2869	prep	in,increase
R496	T2914	T2911	punct	-,outs
R497	T2873	T2874	compound	ES,cells
R498	T2874	T2872	pobj	cells,in
R499	T2915	T2911	prep	in,outs
R5	T443	T441	punct	-,ASAP
R50	T492	T493	advcl	informative,is
R500	T2875	T2866	advmod	clearly,is
R501	T2876	T2877	det	an,goal
R502	T2877	T2866	attr	goal,is
R503	T2916	T2915	pobj	animals,in
R504	T2878	T2877	amod	important,goal
R505	T2917	T2918	punct	(,6
R506	T2879	T2866	punct	.,is
R507	T2918	T2898	parataxis	6,provide
R508	T2881	T2882	prep	In,expedite
R509	T2883	T2881	pobj	addition,In
R51	T494	T493	punct	", ",is
R510	T2884	T2882	punct	", ",expedite
R511	T2885	T2886	amod	efficient,methods
R512	T2919	T2918	punct	),6
R513	T2886	T2882	nsubj	methods,expedite
R514	T2887	T2886	prep	for,methods
R515	T2888	T2889	compound	gene,targeting
R516	T2920	T2898	punct	.,provide
R517	T2889	T2887	pobj	targeting,for
R518	T2890	T2889	prep	in,targeting
R519	T2922	T2923	advmod	However,requires
R52	T495	T496	det	this,approach
R520	T2891	T2890	pobj	fibroblasts,in
R521	T2892	T2882	aux	could,expedite
R522	T2924	T2923	punct	", ",requires
R523	T2893	T2894	amod	phenotypic,analyses
R524	T2894	T2882	dobj	analyses,expedite
R525	T2895	T2882	punct	.,expedite
R526	T2897	T2898	advmod	Indeed,provide
R527	T2925	T2923	csubj	modeling,requires
R528	T2899	T2898	punct	", ",provide
R529	T2926	T2927	advmod	most,mutations
R53	T496	T493	nsubj	approach,is
R530	T2900	T2898	nsubj	siRNAs,provide
R531	T2901	T2900	prep	in,siRNAs
R532	T2902	T2901	pobj	fibroblasts,in
R533	T2927	T2925	dobj	mutations,modeling
R534	T2903	T2898	advmod	often,provide
R535	T2904	T2905	det	a,out
R536	T2905	T2898	dobj	out,provide
R537	T2906	T2905	amod	faster,out
R538	T2928	T2929	npadvmod	disease,associated
R539	T2929	T2927	amod	associated,mutations
R54	T497	T493	acomp	inefficient,is
R540	T2930	T2929	punct	-,associated
R541	T2931	T2923	xcomp	generating,requires
R542	T2932	T2933	amod	subtle,mutations
R543	T3013	T3011	dobj	process,using
R544	T2933	T2931	dobj	mutations,generating
R545	T3014	T3015	nummod	two,step
R546	T3015	T3013	compound	step,process
R547	T3016	T3015	punct	-,step
R548	T2934	T2933	punct	", ",mutations
R549	T3017	T3006	punct	: ,replaced
R55	T498	T497	punct	", ",inefficient
R550	T3018	T3019	det	the,gene
R551	T2935	T2933	cc	not,mutations
R552	T3019	T3006	nsubjpass	gene,replaced
R553	T3020	T3019	prep	of,gene
R554	T3021	T3020	pobj	interest,of
R555	T2936	T2937	compound	knock,outs
R556	T3022	T3006	auxpass	is,replaced
R557	T3023	T3006	advmod	first,replaced
R558	T3024	T3006	agent	by,replaced
R559	T2937	T2933	conj	outs,mutations
R56	T499	T497	conj	slow,inefficient
R560	T3025	T3026	det	a,cassette
R561	T3026	T3024	pobj	cassette,by
R562	T3027	T3026	compound	selection,cassette
R563	T3028	T3026	acl	flanked,cassette
R564	T3029	T3028	agent	by,flanked
R565	T3030	T3031	compound	recombinase,sites
R566	T2938	T2937	punct	-,outs
R567	T3031	T3029	pobj	sites,by
R568	T3032	T3031	compound	target,sites
R569	T3033	T3034	punct	(,sites
R57	T500	T499	cc	and,slow
R570	T2939	T2937	cc	or,outs
R571	T3034	T3031	parataxis	sites,sites
R572	T3035	T3034	advmod	e.g.,sites
R573	T3036	T3034	compound	loxP,sites
R574	T2940	T2941	amod	reduced,expression
R575	T3037	T3034	prep	for,sites
R576	T3038	T3039	compound	Cre,recombinase
R577	T3039	T3037	pobj	recombinase,for
R578	T2941	T2942	compound	expression,alleles
R579	T3040	T3034	punct	", ",sites
R58	T501	T499	conj	expensive,slow
R580	T2942	T2937	conj	alleles,outs
R581	T3041	T3042	aux	to,create
R582	T3042	T3034	advcl	create,sites
R583	T2943	T2923	punct	.,requires
R584	T3043	T3044	det	a,locus
R585	T3044	T3042	dobj	locus,create
R586	T3045	T3044	punct	‘,locus
R587	T2945	T2946	csubj	Targeting,is
R588	T3046	T3044	amod	floxed,locus
R589	T3047	T3044	punct	’,locus
R59	T502	T493	punct	.,is
R590	T3048	T3034	punct	),sites
R591	T3049	T3006	punct	.,replaced
R592	T2947	T2948	compound	point,mutations
R593	T3051	T3052	advmod	Then,removes
R594	T3053	T3052	punct	", ",removes
R595	T2948	T2945	dobj	mutations,Targeting
R596	T2949	T2945	prep	in,Targeting
R597	T3054	T3055	npadvmod	Cre,mediated
R598	T3055	T3057	amod	mediated,recombination
R599	T3056	T3055	punct	-,mediated
R6	T444	T441	punct	: ,ASAP
R60	T504	T505	aux	To,facilitate
R600	T2950	T2949	pobj	fibroblasts,in
R601	T3057	T3052	nsubj	recombination,removes
R602	T3058	T3057	prep	in,recombination
R603	T3059	T3060	det	the,presence
R604	T2951	T2945	prep	by,Targeting
R605	T3060	T3058	pobj	presence,in
R606	T3061	T3060	prep	of,presence
R607	T3062	T3063	det	a,cassette
R608	T2952	T2953	amod	homologous,recombination
R609	T3063	T3061	pobj	cassette,of
R61	T505	T506	advcl	facilitate,developed
R610	T3064	T3063	acl	containing,cassette
R611	T2953	T2951	pobj	recombination,by
R612	T3065	T3066	det	a,allele
R613	T3066	T3064	dobj	allele,containing
R614	T3067	T3066	amod	floxed,allele
R615	T3068	T3066	amod	mutant,allele
R616	T3069	T3070	det	the,sequence
R617	T3070	T3052	dobj	sequence,removes
R618	T2954	T2955	advmod	extremely,inefficient
R619	T3071	T3070	amod	resident,sequence
R62	T507	T505	dobj	targeting,facilitate
R620	T3072	T3052	cc	and,removes
R621	T3073	T3052	conj	inserts,removes
R622	T3074	T3075	det	the,gene
R623	T2955	T2946	acomp	inefficient,is
R624	T3075	T3073	dobj	gene,inserts
R625	T3076	T3075	amod	mutant,gene
R626	T3077	T3078	punct	(,7
R627	T2956	T2946	punct	", ",is
R628	T3078	T3073	parataxis	7,inserts
R629	T3079	T3078	punct	),7
R63	T508	T505	prep	at,facilitate
R630	T3080	T3052	punct	.,removes
R631	T2957	T2946	cc	and,is
R632	T3082	T3083	advmod	Previously,prevented
R633	T2958	T2959	csubjpass	targeting,required
R634	T2959	T2946	conj	required,is
R635	T3084	T3083	punct	", ",prevented
R636	T3085	T3086	amod	technical,difficulties
R637	T3086	T3083	nsubj	difficulties,prevented
R638	T3087	T3083	aux	have,prevented
R639	T2960	T2961	det	both,alleles
R64	T509	T510	det	the,locus
R640	T3088	T3083	dobj	RMCE,prevented
R641	T3089	T3083	prep	from,prevented
R642	T3090	T3091	auxpass	being,applied
R643	T2961	T2958	dobj	alleles,targeting
R644	T3091	T3089	pcomp	applied,from
R645	T3092	T3091	advmod	routinely,applied
R646	T3093	T3094	aux	to,generate
R647	T2962	T2959	auxpass	is,required
R648	T3094	T3091	advcl	generate,applied
R649	T3095	T3096	amod	mutant,mice
R65	T510	T508	pobj	locus,at
R650	T3096	T3094	dobj	mice,generate
R651	T2963	T2964	aux	to,reveal
R652	T3097	T3083	punct	.,prevented
R653	T2964	T2959	advcl	reveal,required
R654	T3099	T3100	prep	For,were
R655	T3101	T3099	pobj	example,For
R656	T2965	T2966	det	the,phenotype
R657	T3102	T3100	punct	", ",were
R658	T3103	T3100	nsubj	exchanges,were
R659	T3104	T3103	acl	using,exchanges
R66	T511	T510	compound	p53,locus
R660	T2966	T2964	dobj	phenotype,reveal
R661	T3105	T3104	dobj	cassettes,using
R662	T3106	T3104	prep	with,using
R663	T3107	T3108	advmod	directly,repeated
R664	T2967	T2966	prep	of,phenotype
R665	T3108	T3109	amod	repeated,sites
R666	T3109	T3106	pobj	sites,with
R667	T3110	T3109	compound	loxP,sites
R668	T3111	T3100	acomp	inefficient,were
R669	T3112	T3113	mark	because,dominated
R67	T512	T506	punct	", ",developed
R670	T3113	T3100	advcl	dominated,were
R671	T2968	T2969	amod	recessive,autosomal
R672	T3114	T3113	nsubj	excisions,dominated
R673	T3115	T3116	det	the,exchanges
R674	T3116	T3113	dobj	exchanges,dominated
R675	T2969	T2970	amod	autosomal,mutations
R676	T3117	T3116	amod	intended,exchanges
R677	T3118	T3119	punct	(,8
R678	T2970	T2967	pobj	mutations,of
R679	T2971	T2959	punct	.,required
R68	T513	T506	nsubj	we,developed
R680	T3119	T3113	parataxis	8,dominated
R681	T2973	T2974	advmod	Here,report
R682	T3120	T3119	punct	),8
R683	T3121	T3100	punct	.,were
R684	T3123	T3124	compound	loxP,sites
R685	T2975	T2974	nsubj	we,report
R686	T3124	T3125	nsubjpass	sites,generated
R687	T3126	T3124	prep	with,sites
R688	T2976	T2977	det	an,approach
R689	T3127	T3128	amod	different,sequences
R69	T514	T515	det	an,method
R690	T3128	T3126	pobj	sequences,with
R691	T2977	T2974	dobj	approach,report
R692	T3129	T3125	auxpass	were,generated
R693	T3130	T3131	aux	to,overcome
R694	T2978	T2979	dep	that,enables
R695	T3131	T3125	advcl	overcome,generated
R696	T3132	T3133	det	this,problem
R697	T3133	T3131	dobj	problem,overcome
R698	T2979	T2977	relcl	enables,approach
R699	T3134	T3125	punct	", ",generated
R7	T445	T446	det	a,method
R70	T515	T506	dobj	method,developed
R700	T3135	T3125	cc	but,generated
R701	T3136	T3137	det	these,sites
R702	T2980	T2981	advmod	highly,efficient
R703	T3137	T3138	nsubj	sites,underwent
R704	T3138	T3125	conj	underwent,generated
R705	T3139	T3138	advmod	also,underwent
R706	T2981	T2982	amod	efficient,targeting
R707	T2982	T2979	dobj	targeting,enables
R708	T3140	T3141	amod	intramolecular,recombination
R709	T3141	T3138	dobj	recombination,underwent
R71	T516	T515	amod	improved,method
R710	T2983	T2979	prep	at,enables
R711	T3142	T3138	punct	", ",underwent
R712	T3143	T3138	advcl	making,underwent
R713	T2984	T2985	det	the,locus
R714	T3144	T3145	nsubj	RMCE,efficient
R715	T3145	T3143	ccomp	efficient,making
R716	T3146	T3147	advmod	only,contained
R717	T3147	T3143	advcl	contained,making
R718	T3148	T3147	mark	if,contained
R719	T2985	T2983	pobj	locus,at
R72	T517	T518	npadvmod	Recombinase,Mediated
R720	T3149	T3150	det	the,cassette
R721	T3150	T3147	nsubj	cassette,contained
R722	T3151	T3150	compound	replacement,cassette
R723	T3152	T3153	det	a,marker
R724	T2986	T2985	compound	p53,locus
R725	T3153	T3147	dobj	marker,contained
R726	T3154	T3153	acl	enabling,marker
R727	T2987	T2979	prep	in,enables
R728	T3155	T3154	dobj	selection,enabling
R729	T3156	T3155	prep	of,selection
R73	T518	T520	amod	Mediated,Exchange
R730	T3157	T3158	det	the,recombinant
R731	T3158	T3156	pobj	recombinant,of
R732	T2988	T2989	preconj	both,cells
R733	T3159	T3158	amod	desired,recombinant
R734	T3160	T3161	punct	(,7
R735	T2989	T2987	pobj	cells,in
R736	T3161	T3147	parataxis	7,contained
R737	T3162	T3161	punct	",",7
R738	T3163	T3161	appos	9,7
R739	T2990	T2989	compound	ES,cells
R74	T519	T518	punct	-,Mediated
R740	T3164	T3165	punct	–,12
R741	T3165	T3163	prep	12,9
R742	T2991	T2989	cc	and,cells
R743	T3166	T3161	punct	),7
R744	T3167	T3138	punct	.,underwent
R745	T2992	T2989	conj	fibroblasts,cells
R746	T3169	T3170	advmod	However,necessitates
R747	T3171	T3170	punct	", ",necessitates
R748	T3172	T3170	nsubj	interference,necessitates
R749	T2993	T2974	punct	.,report
R75	T520	T515	nmod	Exchange,method
R750	T3173	T3172	acl	resulting,interference
R751	T3174	T3173	prep	from,resulting
R752	T2995	T2996	npadvmod	Recombinase,Mediated
R753	T3175	T3174	pobj	expression,from
R754	T3176	T3175	prep	of,expression
R755	T3177	T3178	det	the,marker
R756	T3178	T3176	pobj	marker,of
R757	T2996	T2998	amod	Mediated,Exchange
R758	T3179	T3178	compound	selection,marker
R759	T3180	T3178	cc	and,marker
R76	T521	T520	nmod	Cassette,Exchange
R760	T3181	T3182	det	the,gene
R761	T2997	T2996	punct	-,Mediated
R762	T3182	T3178	conj	gene,marker
R763	T3183	T3182	amod	endogenous,gene
R764	T3184	T3185	punct	(,13
R765	T2998	T3000	nmod	Exchange,approaches
R766	T3185	T3173	parataxis	13,resulting
R767	T3186	T3185	punct	),13
R768	T3187	T3170	dobj	strategies,necessitates
R769	T3188	T3189	aux	to,remove
R77	T522	T520	punct	(,Exchange
R770	T3189	T3187	advcl	remove,strategies
R771	T3190	T3191	det	the,gene
R772	T3191	T3189	dobj	gene,remove
R773	T2999	T2998	nmod	Cassette,Exchange
R774	T3192	T3191	amod	selectable,gene
R775	T3193	T3170	punct	.,necessitates
R776	T3000	T3004	nsubjpass	approaches,developed
R777	T3195	T3196	advmod	Together,indicate
R778	T3197	T3196	punct	", ",indicate
R779	T3001	T2998	punct	(,Exchange
R78	T523	T520	appos	RMCE,Exchange
R780	T3198	T3199	amod	previous,studies
R781	T3199	T3196	nsubj	studies,indicate
R782	T3200	T3201	mark	that,requires
R783	T3002	T2998	appos	RMCE,Exchange
R784	T3201	T3196	ccomp	requires,indicate
R785	T3202	T3203	det	an,RMCE
R786	T3003	T3000	punct	),approaches
R787	T3203	T3201	nsubj	RMCE,requires
R788	T3204	T3203	amod	optimal,RMCE
R789	T3205	T3206	punct	(,i
R79	T524	T515	punct	),method
R790	T3004	T3006	ccomp	developed,replaced
R791	T3206	T3207	meta	i,sites
R792	T3207	T3201	dobj	sites,requires
R793	T3208	T3206	punct	),i
R794	T3005	T3004	auxpass	were,developed
R795	T3209	T3207	amod	inverted,sites
R796	T3007	T3008	aux	to,improve
R797	T3210	T3207	amod	heterologous,sites
R798	T3211	T3207	compound	loxP,sites
R799	T3212	T3207	acl	diverging,sites
R8	T446	T441	appos	method,ASAP
R80	T525	T506	punct	.,developed
R800	T3213	T3212	prep	by,diverging
R801	T3214	T3215	quantmod	at,2
R802	T3215	T3217	nummod	2,nt
R803	T3008	T3004	advcl	improve,developed
R804	T3216	T3214	advmod	least,at
R805	T3217	T3213	pobj	nt,by
R806	T3218	T3219	aux	to,maximize
R807	T3009	T3010	amod	targeting,efficiency
R808	T3219	T3207	advcl	maximize,sites
R809	T3220	T3221	det	the,efficiency
R81	T527	T528	poss	Our,approach
R810	T3221	T3219	dobj	efficiency,maximize
R811	T3010	T3008	dobj	efficiency,improve
R812	T3222	T3221	prep	of,efficiency
R813	T3223	T3222	pobj	exchange,of
R814	T3011	T3008	advcl	using,improve
R815	T3012	T3013	det	a,process
R816	T3224	T3207	cc	and,sites
R817	T3331	T3330	punct	–,Thymidine
R818	T3332	T3328	compound	Kinase,gene
R819	T3225	T3226	punct	(,ii
R82	T528	T529	nsubj	approach,enables
R820	T3226	T3227	meta	ii,cassette
R821	T3333	T3328	compound	fusion,gene
R822	T3227	T3207	conj	cassette,sites
R823	T3228	T3226	punct	),ii
R824	T3229	T3227	det	an,cassette
R825	T3230	T3227	compound	expression,cassette
R826	T3231	T3227	acl	enabling,cassette
R827	T3232	T3233	preconj	both,selection
R828	T3334	T3328	acl	used,gene
R829	T3233	T3231	dobj	selection,enabling
R83	T530	T531	amod	efficient,targeting
R830	T3234	T3233	amod	positive,selection
R831	T3335	T3336	advmod	most,frequently
R832	T3235	T3236	aux	to,identify
R833	T3236	T3231	advcl	identify,enabling
R834	T3237	T3238	det	the,recombinant
R835	T3336	T3334	advmod	frequently,used
R836	T3238	T3236	dobj	recombinant,identify
R837	T3239	T3238	amod	initial,recombinant
R838	T3240	T3231	cc	and,enabling
R839	T3337	T3334	prep	for,used
R84	T531	T529	dobj	targeting,enables
R840	T3241	T3242	advmod	then,selection
R841	T3242	T3231	conj	selection,enabling
R842	T3243	T3242	amod	negative,selection
R843	T3244	T3245	aux	to,obtain
R844	T3338	T3339	amod	positive,negative
R845	T3245	T3242	advcl	obtain,selection
R846	T3246	T3247	det	a,allele
R847	T3247	T3245	dobj	allele,obtain
R848	T3339	T3341	nmod	negative,selection
R849	T3248	T3249	punct	‘,free
R85	T532	T531	prep	in,targeting
R850	T3249	T3247	amod	free,allele
R851	T3340	T3339	punct	/,negative
R852	T3250	T3249	npadvmod	marker,free
R853	T3251	T3249	punct	-,free
R854	T3252	T3249	punct	’,free
R855	T3341	T3337	pobj	selection,for
R856	T3253	T3247	amod	mutant,allele
R857	T3254	T3255	punct	(,14
R858	T3255	T3242	parataxis	14,selection
R859	T3342	T3341	prep	in,selection
R86	T533	T534	compound	ES,cells
R860	T3256	T3255	punct	),14
R861	T3257	T3196	punct	.,indicate
R862	T3343	T3342	pobj	RMCE,in
R863	T3259	T3260	amod	Most,experiments
R864	T3260	T3262	nsubjpass	experiments,performed
R865	T3261	T3260	compound	RMCE,experiments
R866	T3344	T3325	punct	", ",leads
R867	T3263	T3262	aux	have,performed
R868	T3264	T3262	auxpass	been,performed
R869	T3345	T3325	prep	to,leads
R87	T534	T532	pobj	cells,in
R870	T3265	T3262	prep	at,performed
R871	T3266	T3267	amod	random,sites
R872	T3267	T3265	pobj	sites,at
R873	T3346	T3347	compound	mouse,sterility
R874	T3268	T3267	prep	in,sites
R875	T3269	T3270	amod	somatic,cell
R876	T3270	T3271	compound	cell,lines
R877	T3271	T3268	pobj	lines,in
R878	T3272	T3262	punct	.,performed
R879	T3347	T3345	pobj	sterility,to
R88	T535	T536	aux	to,facilitate
R880	T3274	T3275	advmod	Only,mice
R881	T3275	T3279	nsubjpass	mice,reported
R882	T3276	T3275	det	a,mice
R883	T3348	T3325	punct	", ",leads
R884	T3277	T3275	amod	few,mice
R885	T3278	T3275	amod	mutant,mice
R886	T3349	T3350	mark	so,performed
R887	T3280	T3275	acl	generated,mice
R888	T3281	T3280	agent	by,generated
R889	T3282	T3281	pobj	RMCE,by
R89	T536	T529	xcomp	facilitate,enables
R890	T3350	T3325	advcl	performed,leads
R891	T3283	T3282	prep	in,RMCE
R892	T3284	T3285	compound	ES,cells
R893	T3351	T3350	mark	that,performed
R894	T3285	T3283	pobj	cells,in
R895	T3286	T3279	aux	have,reported
R896	T3287	T3279	auxpass	been,reported
R897	T3288	T3279	punct	", ",reported
R898	T3352	T3350	nsubjpass	exchanges,performed
R899	T3289	T3279	cc	but,reported
R9	T447	T446	nmod	gene,method
R90	T537	T538	det	the,production
R900	T3290	T3291	det	the,RMCE
R901	T3353	T3350	aux	can,performed
R902	T3291	T3292	nsubj	RMCE,introduced
R903	T3292	T3279	conj	introduced,reported
R904	T3354	T3350	advmod	only,performed
R905	T3293	T3292	advmod	systematically,introduced
R906	T3294	T3295	det	a,marker
R907	T3295	T3292	dobj	marker,introduced
R908	T3296	T3295	amod	selectable,marker
R909	T3355	T3350	auxpass	be,performed
R91	T538	T536	dobj	production,facilitate
R910	T3297	T3298	punct	(,15–17
R911	T3298	T3292	parataxis	15–17,introduced
R912	T3299	T3298	punct	),15–17
R913	T3356	T3350	prep	in,performed
R914	T3300	T3292	punct	", ",introduced
R915	T3301	T3292	cc	or,introduced
R916	T3357	T3358	compound	ES,cells
R917	T3302	T3292	punct	", ",introduced
R918	T3303	T3304	advmod	when,tested
R919	T3358	T3356	pobj	cells,in
R92	T539	T538	prep	of,production
R920	T3304	T3305	advcl	tested,proved
R921	T3359	T3360	punct	(,16
R922	T3305	T3292	conj	proved,introduced
R923	T3360	T3325	parataxis	16,leads
R924	T3306	T3304	prep	without,tested
R925	T3307	T3308	det	an,marker
R926	T3308	T3306	pobj	marker,without
R927	T3309	T3308	amod	incoming,marker
R928	T3310	T3305	punct	", ",proved
R929	T3311	T3305	oprd	inefficient,proved
R93	T540	T541	amod	mutant,mice
R930	T3312	T3313	punct	(,12
R931	T3361	T3360	punct	),16
R932	T3313	T3305	parataxis	12,proved
R933	T3314	T3313	punct	),12
R934	T3315	T3292	punct	.,introduced
R935	T3362	T3325	punct	.,leads
R936	T3317	T3318	det	A,report
R937	T3364	T3365	det	The,strategy
R938	T3318	T3320	nsubj	report,disclosed
R939	T3319	T3318	amod	recent,report
R94	T541	T539	pobj	mice,of
R940	T3321	T3322	det	an,problem
R941	T3365	T3367	nsubj	strategy,relies
R942	T3322	T3320	dobj	problem,disclosed
R943	T3323	T3322	amod	additional,problem
R944	T3324	T3325	punct	: ,leads
R945	T3366	T3365	compound	RMCE,strategy
R946	T3327	T3328	det	the,gene
R947	T3368	T3365	acl	presented,strategy
R948	T3328	T3325	nsubj	gene,leads
R949	T3329	T3330	compound	Hygromycin,Thymidine
R95	T542	T529	punct	.,enables
R950	T3330	T3332	compound	Thymidine,Kinase
R951	T3369	T3368	advmod	here,presented
R952	T3370	T3367	prep	on,relies
R953	T3371	T3372	det	the,use
R954	T3372	T3370	pobj	use,on
R955	T3437	T3435	nsubjpass	it,Adapted
R956	T3373	T3372	amod	integrated,use
R957	T3438	T3435	auxpass	is,Adapted
R958	T3439	T3435	prep	for,Adapted
R959	T3374	T3372	prep	of,use
R96	T544	T545	cc	But,Adapted
R960	T3440	T3439	pobj	targeting,for
R961	T3375	T3376	amod	inverted,sites
R962	T3441	T3440	prep	in,targeting
R963	T3442	T3443	amod	Somatic,cells
R964	T3443	T3441	pobj	cells,in
R965	T3376	T3374	pobj	sites,of
R966	T3444	T3445	aux	to,Accelerate
R967	T3445	T3435	advcl	Accelerate,Adapted
R968	T3377	T3376	amod	heterologous,sites
R969	T3446	T3445	dobj	Phenotyping,Accelerate
R97	T546	T547	advmod	more,importantly
R970	T3447	T3446	punct	(,Phenotyping
R971	T3448	T3446	appos	ASAP,Phenotyping
R972	T3449	T3435	punct	),Adapted
R973	T3378	T3376	compound	loxP,sites
R974	T3450	T3435	punct	.,Adapted
R975	T3379	T3372	punct	", ",use
R976	T3452	T3453	mark	Because,relies
R977	T3453	T3455	advcl	relies,applied
R978	T3454	T3453	nsubj	it,relies
R979	T3456	T3453	prep	on,relies
R98	T547	T545	advmod	importantly,Adapted
R980	T3457	T3458	advmod	very,principles
R981	T3380	T3381	det	a,marker
R982	T3458	T3456	pobj	principles,on
R983	T3459	T3458	amod	general,principles
R984	T3460	T3455	punct	", ",applied
R985	T3381	T3372	conj	marker,use
R986	T3461	T3462	compound	RMCE,ASAP
R987	T3462	T3455	nsubjpass	ASAP,applied
R988	T3463	T3462	punct	-,ASAP
R989	T3382	T3383	amod	positive,negative
R99	T548	T545	punct	", ",Adapted
R990	T3464	T3455	aux	could,applied
R991	T3465	T3455	auxpass	be,applied
R992	T3466	T3455	prep	to,applied
R993	T3383	T3385	nmod	negative,selection
R994	T3467	T3468	det	any,locus
R995	T3468	T3466	pobj	locus,to
R996	T3469	T3468	prep	of,locus
R997	T3384	T3383	punct	/,negative
R998	T3470	T3469	pobj	interest,of
R999	T3471	T3455	punct	.,applied

PMC:1540739 JSONTXT 9 Projects

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PMC:1540739 JSON TXT 9 Projects