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PMC:1540739 / 1130-21382 JSONTXT 5 Projects

Annnotations TAB TSV DIC JSON TextAE

Id Subject Object Predicate Lexical cue
T2571 13-16 NN denotes p53
T2572 17-19 VBZ denotes is
T2573 20-23 CD denotes one
T2574 24-26 IN denotes of
T2575 27-30 DT denotes the
T2577 31-35 RBS denotes most
T2578 36-42 RB denotes highly
T2579 43-51 JJ denotes analyzed
T2576 52-60 NN denotes proteins
T2580 61-64 IN denotes for
T2581 65-68 DT denotes the
T2583 69-73 JJ denotes past
T2584 74-76 CD denotes 25
T2582 77-82 NNS denotes years
T2585 82-83 . denotes .
T2586 83-245 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 84-91 NNS denotes Studies
T2589 92-94 IN denotes in
T2590 95-103 VBN denotes cultured
T2591 104-109 NNS denotes cells
T2592 109-111 , denotes ,
T2593 111-116 RB denotes often
T2594 117-124 VBG denotes relying
T2595 125-127 IN denotes on
T2596 128-131 DT denotes the
T2597 132-144 NN denotes transfection
T2598 145-147 IN denotes of
T2599 148-156 NNS denotes plasmids
T2600 157-167 VBG denotes expressing
T2601 168-175 JJ denotes various
T2603 176-179 NN denotes p53
T2602 180-187 NNS denotes mutants
T2604 187-189 , denotes ,
T2605 189-193 VBD denotes have
T2588 194-205 VBN denotes established
T2606 206-212 NNS denotes models
T2607 213-215 TO denotes to
T2608 216-223 VB denotes explain
T2609 224-227 WRB denotes how
T2611 228-231 NN denotes p53
T2612 232-234 VBZ denotes is
T2610 235-244 VBN denotes regulated
T2613 244-245 . denotes .
T2614 245-435 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 246-248 IN denotes In
T2617 249-255 JJ denotes recent
T2618 256-261 NNS denotes years
T2619 261-263 , denotes ,
T2620 263-267 DT denotes some
T2621 268-270 IN denotes of
T2622 271-276 DT denotes these
T2623 277-283 NNS denotes models
T2624 284-288 VBD denotes were
T2616 289-295 VBN denotes tested
T2625 296-298 FW denotes in
T2626 299-303 FW denotes vivo
T2627 304-306 IN denotes by
T2628 307-316 VBG denotes targeting
T2629 317-323 JJ denotes subtle
T2630 324-333 NNS denotes mutations
T2631 334-336 IN denotes at
T2632 337-340 DT denotes the
T2634 341-344 NN denotes p53
T2633 345-350 NN denotes locus
T2635 351-356 VBG denotes using
T2636 357-367 JJ denotes homologous
T2637 368-381 NN denotes recombination
T2638 382-384 IN denotes in
T2639 385-394 JJ denotes embryonic
T2640 395-399 NN denotes stem
T2642 400-401 -LRB- denotes (
T2643 401-403 NN denotes ES
T2644 403-404 -RRB- denotes )
T2641 405-410 NNS denotes cells
T2645 411-413 TO denotes to
T2646 414-422 VB denotes generate
T2647 423-429 JJ denotes mutant
T2648 430-434 NNS denotes mice
T2649 434-435 . denotes .
T2650 435-638 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 436-439 DT denotes The
T2652 440-448 NN denotes strength
T2654 449-451 IN denotes of
T2655 452-456 DT denotes this
T2656 457-465 NN denotes approach
T2653 466-468 VBZ denotes is
T2657 469-473 IN denotes that
T2659 474-483 NNS denotes mutations
T2660 484-487 VBP denotes are
T2658 488-494 VBN denotes tested
T2661 495-497 IN denotes in
T2662 498-499 DT denotes a
T2664 500-507 JJ denotes genomic
T2663 508-515 NN denotes setting
T2665 516-519 CC denotes and
T2666 520-529 VBN denotes expressed
T2667 530-534 IN denotes from
T2668 535-538 DT denotes the
T2670 539-549 JJ denotes endogenous
T2669 550-558 NN denotes promoter
T2671 558-560 , denotes ,
T2672 560-568 VBG denotes ensuring
T2673 569-582 JJ denotes physiological
T2675 583-593 NN denotes expression
T2674 594-600 NNS denotes levels
T2676 601-604 CC denotes and
T2677 605-612 JJ denotes correct
T2679 613-619 JJ denotes spatio
T2681 619-620 HYPH denotes -
T2680 620-628 JJ denotes temporal
T2678 629-637 NNS denotes profiles
T2682 637-638 . denotes .
T2683 638-904 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 639-641 IN denotes As
T2686 642-653 JJ denotes significant
T2687 654-665 NNS denotes differences
T2688 666-673 IN denotes between
T2689 674-684 NNS denotes phenotypes
T2690 685-689 IN denotes from
T2691 690-698 VBN denotes targeted
T2693 699-702 NN denotes p53
T2692 703-710 NNS denotes mutants
T2694 711-713 FW denotes in
T2695 714-718 FW denotes vivo
T2696 719-722 CC denotes and
T2697 723-735 NN denotes transfection
T2698 736-740 NNS denotes data
T2699 741-745 VBD denotes were
T2685 746-754 VBN denotes observed
T2701 755-756 -LRB- denotes [
T2703 756-760 FW denotes e.g.
T2704 761-765 NNS denotes Refs
T2705 766-767 -LRB- denotes (
T2702 767-768 CD denotes 1
T2706 768-769 SYM denotes –
T2707 769-770 CD denotes 5
T2708 770-771 -RRB- denotes )
T2709 771-772 -RRB- denotes ]
T2710 772-774 , denotes ,
T2711 774-778 RBR denotes more
T2712 779-787 VBN denotes targeted
T2713 788-797 NNS denotes mutations
T2700 798-802 VBP denotes need
T2714 803-805 TO denotes to
T2716 806-808 VB denotes be
T2715 809-818 VBN denotes generated
T2717 819-822 CC denotes and
T2718 823-831 VBN denotes analyzed
T2719 832-834 IN denotes in
T2720 835-843 JJ denotes multiple
T2721 844-851 NNS denotes tissues
T2722 852-854 TO denotes to
T2723 855-864 VB denotes formulate
T2724 865-869 RBR denotes more
T2725 870-878 JJ denotes accurate
T2726 879-885 NNS denotes models
T2727 886-888 IN denotes of
T2728 889-892 NN denotes p53
T2729 893-903 NN denotes regulation
T2730 903-904 . denotes .
T2731 904-1425 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 905-912 RB denotes However
T2734 912-914 , denotes ,
T2735 914-919 VBG denotes using
T2736 920-930 JJ denotes homologous
T2737 931-944 NN denotes recombination
T2738 945-947 IN denotes in
T2739 948-950 NN denotes ES
T2740 951-956 NNS denotes cells
T2741 957-959 TO denotes to
T2742 960-968 VB denotes generate
T2743 969-975 JJ denotes mutant
T2744 976-980 NNS denotes mice
T2733 981-983 VBZ denotes is
T2745 984-986 DT denotes an
T2747 987-998 JJ denotes inefficient
T2748 998-1000 , denotes ,
T2749 1000-1004 JJ denotes slow
T2750 1005-1008 CC denotes and
T2751 1009-1018 JJ denotes expensive
T2746 1019-1025 NN denotes method
T2752 1026-1033 IN denotes because
T2754 1034-1035 -LRB- denotes (
T2755 1035-1036 LS denotes i
T2756 1036-1037 -RRB- denotes )
T2757 1038-1048 JJ denotes homologous
T2758 1049-1062 NN denotes recombination
T2759 1063-1072 RB denotes typically
T2753 1073-1079 VBZ denotes occurs
T2760 1080-1082 IN denotes at
T2761 1083-1086 JJ denotes low
T2762 1087-1096 NN denotes frequency
T2763 1097-1099 IN denotes in
T2764 1100-1102 NN denotes ES
T2765 1103-1108 NNS denotes cells
T2766 1108-1110 , denotes ,
T2767 1110-1119 VBG denotes requiring
T2768 1120-1133 JJ denotes sophisticated
T2770 1134-1143 NN denotes selection
T2769 1144-1151 NNS denotes schemes
T2771 1152-1155 CC denotes and
T2772 1156-1165 NN denotes screening
T2773 1166-1168 IN denotes of
T2774 1169-1177 NNS denotes hundreds
T2775 1178-1180 IN denotes of
T2776 1181-1187 NNS denotes clones
T2777 1188-1190 TO denotes to
T2778 1191-1199 VB denotes identify
T2779 1200-1203 DT denotes the
T2781 1204-1211 JJ denotes desired
T2780 1212-1218 NN denotes mutant
T2782 1218-1219 : denotes ;
T2783 1220-1221 -LRB- denotes (
T2784 1221-1223 LS denotes ii
T2786 1223-1224 -RRB- denotes )
T2787 1225-1230 JJ denotes large
T2788 1231-1232 -LRB- denotes (
T2790 1232-1234 CD denotes 15
T2792 1234-1235 SYM denotes –
T2791 1235-1237 CD denotes 20
T2789 1238-1240 NN denotes kb
T2793 1240-1241 -RRB- denotes )
T2785 1242-1250 NNS denotes plasmids
T2795 1250-1252 , denotes ,
T2796 1252-1257 RB denotes often
T2797 1258-1267 JJ denotes difficult
T2798 1268-1270 TO denotes to
T2799 1271-1276 VB denotes clone
T2800 1276-1278 , denotes ,
T2801 1278-1281 VBP denotes are
T2794 1282-1290 VBN denotes required
T2802 1291-1293 TO denotes to
T2803 1294-1302 VB denotes increase
T2804 1303-1312 VBG denotes targeting
T2805 1313-1323 NN denotes efficiency
T2806 1324-1327 CC denotes and
T2807 1328-1329 -LRB- denotes (
T2808 1329-1332 LS denotes iii
T2810 1332-1333 -RRB- denotes )
T2811 1334-1342 VBG denotes breeding
T2812 1343-1347 NNS denotes mice
T2813 1348-1350 IN denotes to
T2814 1351-1363 NN denotes homozygosity
T2815 1364-1367 CC denotes and
T2816 1368-1375 VBG denotes housing
T2817 1376-1377 DT denotes a
T2819 1378-1383 NN denotes mouse
T2818 1384-1390 NN denotes colony
T2809 1391-1399 VBP denotes generate
T2820 1400-1407 JJ denotes further
T2821 1408-1414 NNS denotes delays
T2822 1415-1418 CC denotes and
T2823 1419-1424 NNS denotes costs
T2824 1424-1425 . denotes .
T2825 1425-1539 sentence denotes Such limitations make the repeated targeting of a locus a technically daunting and economically impractical task.
T2826 1426-1430 JJ denotes Such
T2827 1431-1442 NNS denotes limitations
T2828 1443-1447 VBP denotes make
T2829 1448-1451 DT denotes the
T2831 1452-1460 VBN denotes repeated
T2830 1461-1470 NN denotes targeting
T2833 1471-1473 IN denotes of
T2834 1474-1475 DT denotes a
T2835 1476-1481 NN denotes locus
T2836 1482-1483 DT denotes a
T2837 1484-1495 RB denotes technically
T2838 1496-1504 VBG denotes daunting
T2839 1505-1508 CC denotes and
T2840 1509-1521 RB denotes economically
T2841 1522-1533 JJ denotes impractical
T2832 1534-1538 NN denotes task
T2842 1538-1539 . denotes .
T2843 1539-1652 sentence denotes Improvements in current technologies are needed to enable such analyses to be applied to the p53 or other genes.
T2844 1540-1552 NNS denotes Improvements
T2846 1553-1555 IN denotes in
T2847 1556-1563 JJ denotes current
T2848 1564-1576 NNS denotes technologies
T2849 1577-1580 VBP denotes are
T2845 1581-1587 VBN denotes needed
T2850 1588-1590 TO denotes to
T2851 1591-1597 VB denotes enable
T2852 1598-1602 JJ denotes such
T2853 1603-1611 NNS denotes analyses
T2854 1612-1614 TO denotes to
T2856 1615-1617 VB denotes be
T2855 1618-1625 VBN denotes applied
T2857 1626-1628 IN denotes to
T2858 1629-1632 DT denotes the
T2859 1633-1636 NN denotes p53
T2860 1637-1639 CC denotes or
T2861 1640-1645 JJ denotes other
T2862 1646-1651 NNS denotes genes
T2863 1651-1652 . denotes .
T2864 1652-1746 sentence denotes Developing methods to increase targeting efficiency in ES cells is clearly an important goal.
T2865 1653-1663 VBG denotes Developing
T2867 1664-1671 NNS denotes methods
T2868 1672-1674 TO denotes to
T2869 1675-1683 VB denotes increase
T2870 1684-1693 VBG denotes targeting
T2871 1694-1704 NN denotes efficiency
T2872 1705-1707 IN denotes in
T2873 1708-1710 NN denotes ES
T2874 1711-1716 NNS denotes cells
T2866 1717-1719 VBZ denotes is
T2875 1720-1727 RB denotes clearly
T2876 1728-1730 DT denotes an
T2878 1731-1740 JJ denotes important
T2877 1741-1745 NN denotes goal
T2879 1745-1746 . denotes .
T2880 1746-1847 sentence denotes In addition, efficient methods for gene targeting in fibroblasts could expedite phenotypic analyses.
T2881 1747-1749 IN denotes In
T2883 1750-1758 NN denotes addition
T2884 1758-1760 , denotes ,
T2885 1760-1769 JJ denotes efficient
T2886 1770-1777 NNS denotes methods
T2887 1778-1781 IN denotes for
T2888 1782-1786 NN denotes gene
T2889 1787-1796 NN denotes targeting
T2890 1797-1799 IN denotes in
T2891 1800-1811 NNS denotes fibroblasts
T2892 1812-1817 MD denotes could
T2882 1818-1826 VB denotes expedite
T2893 1827-1837 JJ denotes phenotypic
T2894 1838-1846 NNS denotes analyses
T2895 1846-1847 . denotes .
T2896 1847-1957 sentence denotes Indeed, siRNAs in fibroblasts often provide a faster read-out than equivalent gene knock-outs in animals (6).
T2897 1848-1854 RB denotes Indeed
T2899 1854-1856 , denotes ,
T2900 1856-1862 NNS denotes siRNAs
T2901 1863-1865 IN denotes in
T2902 1866-1877 NNS denotes fibroblasts
T2903 1878-1883 RB denotes often
T2898 1884-1891 VBP denotes provide
T2904 1892-1893 DT denotes a
T2906 1894-1900 JJR denotes faster
T2907 1901-1905 NN denotes read
T2908 1905-1906 HYPH denotes -
T2905 1906-1909 NN denotes out
T2909 1910-1914 IN denotes than
T2910 1915-1925 JJ denotes equivalent
T2912 1926-1930 NN denotes gene
T2913 1931-1936 NN denotes knock
T2914 1936-1937 HYPH denotes -
T2911 1937-1941 NNS denotes outs
T2915 1942-1944 IN denotes in
T2916 1945-1952 NNS denotes animals
T2917 1953-1954 -LRB- denotes (
T2918 1954-1955 CD denotes 6
T2919 1955-1956 -RRB- denotes )
T2920 1956-1957 . denotes .
T2921 1957-2093 sentence denotes However, modeling most disease-associated mutations requires generating subtle mutations, not knock-outs or reduced expression alleles.
T2922 1958-1965 RB denotes However
T2924 1965-1967 , denotes ,
T2925 1967-1975 VBG denotes modeling
T2926 1976-1980 RBS denotes most
T2928 1981-1988 NN denotes disease
T2930 1988-1989 HYPH denotes -
T2929 1989-1999 JJ denotes associated
T2927 2000-2009 NNS denotes mutations
T2923 2010-2018 VBZ denotes requires
T2931 2019-2029 VBG denotes generating
T2932 2030-2036 JJ denotes subtle
T2933 2037-2046 NNS denotes mutations
T2934 2046-2048 , denotes ,
T2935 2048-2051 CC denotes not
T2936 2052-2057 NN denotes knock
T2938 2057-2058 HYPH denotes -
T2937 2058-2062 NNS denotes outs
T2939 2063-2065 CC denotes or
T2940 2066-2073 VBN denotes reduced
T2941 2074-2084 NN denotes expression
T2942 2085-2092 NNS denotes alleles
T2943 2092-2093 . denotes .
T2944 2093-2285 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 2094-2103 VBG denotes Targeting
T2947 2104-2109 NN denotes point
T2948 2110-2119 NNS denotes mutations
T2949 2120-2122 IN denotes in
T2950 2123-2134 NNS denotes fibroblasts
T2951 2135-2137 IN denotes by
T2952 2138-2148 JJ denotes homologous
T2953 2149-2162 NN denotes recombination
T2946 2163-2165 VBZ denotes is
T2954 2166-2175 RB denotes extremely
T2955 2176-2187 JJ denotes inefficient
T2956 2187-2189 , denotes ,
T2957 2189-2192 CC denotes and
T2958 2193-2202 VBG denotes targeting
T2960 2203-2207 DT denotes both
T2961 2208-2215 NNS denotes alleles
T2962 2216-2218 VBZ denotes is
T2959 2219-2227 VBN denotes required
T2963 2228-2230 TO denotes to
T2964 2231-2237 VB denotes reveal
T2965 2238-2241 DT denotes the
T2966 2242-2251 NN denotes phenotype
T2967 2252-2254 IN denotes of
T2968 2255-2264 JJ denotes recessive
T2969 2265-2274 JJ denotes autosomal
T2970 2275-2284 NNS denotes mutations
T2971 2284-2285 . denotes .
T2972 2285-2403 sentence denotes Here we report an approach that enables highly efficient targeting at the p53 locus in both ES cells and fibroblasts.
T2973 2286-2290 RB denotes Here
T2975 2291-2293 PRP denotes we
T2974 2294-2300 VBP denotes report
T2976 2301-2303 DT denotes an
T2977 2304-2312 NN denotes approach
T2978 2313-2317 WDT denotes that
T2979 2318-2325 VBZ denotes enables
T2980 2326-2332 RB denotes highly
T2981 2333-2342 JJ denotes efficient
T2982 2343-2352 NN denotes targeting
T2983 2353-2355 IN denotes at
T2984 2356-2359 DT denotes the
T2986 2360-2363 NN denotes p53
T2985 2364-2369 NN denotes locus
T2987 2370-2372 IN denotes in
T2988 2373-2377 CC denotes both
T2990 2378-2380 NN denotes ES
T2989 2381-2386 NNS denotes cells
T2991 2387-2390 CC denotes and
T2992 2391-2402 NNS denotes fibroblasts
T2993 2402-2403 . denotes .
T2994 2403-2699 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 2404-2415 NN denotes Recombinase
T2997 2415-2416 HYPH denotes -
T2996 2416-2424 JJ denotes Mediated
T2999 2425-2433 NNP denotes Cassette
T2998 2434-2442 NNP denotes Exchange
T3001 2443-2444 -LRB- denotes (
T3002 2444-2448 NN denotes RMCE
T3003 2448-2449 -RRB- denotes )
T3000 2450-2460 NNS denotes approaches
T3005 2461-2465 VBD denotes were
T3004 2466-2475 VBN denotes developed
T3007 2476-2478 TO denotes to
T3008 2479-2486 VB denotes improve
T3009 2487-2496 VBG denotes targeting
T3010 2497-2507 NN denotes efficiency
T3011 2508-2513 VBG denotes using
T3012 2514-2515 DT denotes a
T3014 2516-2519 CD denotes two
T3016 2519-2520 HYPH denotes -
T3015 2520-2524 NN denotes step
T3013 2525-2532 NN denotes process
T3017 2532-2534 : denotes :
T3018 2534-2537 DT denotes the
T3019 2538-2542 NN denotes gene
T3020 2543-2545 IN denotes of
T3021 2546-2554 NN denotes interest
T3022 2555-2557 VBZ denotes is
T3023 2558-2563 RB denotes first
T3006 2564-2572 VBN denotes replaced
T3024 2573-2575 IN denotes by
T3025 2576-2577 DT denotes a
T3027 2578-2587 NN denotes selection
T3026 2588-2596 NN denotes cassette
T3028 2597-2604 VBN denotes flanked
T3029 2605-2607 IN denotes by
T3030 2608-2619 NN denotes recombinase
T3032 2620-2626 NN denotes target
T3031 2627-2632 NNS denotes sites
T3033 2633-2634 -LRB- denotes (
T3035 2634-2638 FW denotes e.g.
T3036 2639-2643 NN denotes loxP
T3034 2644-2649 NNS denotes sites
T3037 2650-2653 IN denotes for
T3038 2654-2657 NN denotes Cre
T3039 2658-2669 NN denotes recombinase
T3040 2669-2671 , denotes ,
T3041 2671-2673 TO denotes to
T3042 2674-2680 VB denotes create
T3043 2681-2682 DT denotes a
T3045 2683-2684 `` denotes ‘
T3046 2684-2690 JJ denotes floxed
T3047 2690-2691 '' denotes ’
T3044 2692-2697 NN denotes locus
T3048 2697-2698 -RRB- denotes )
T3049 2698-2699 . denotes .
T3050 2699-2859 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 2700-2704 RB denotes Then
T3053 2704-2706 , denotes ,
T3054 2706-2709 NN denotes Cre
T3056 2709-2710 HYPH denotes -
T3055 2710-2718 JJ denotes mediated
T3057 2719-2732 NN denotes recombination
T3058 2733-2735 IN denotes in
T3059 2736-2739 DT denotes the
T3060 2740-2748 NN denotes presence
T3061 2749-2751 IN denotes of
T3062 2752-2753 DT denotes a
T3063 2754-2762 NN denotes cassette
T3064 2763-2773 VBG denotes containing
T3065 2774-2775 DT denotes a
T3067 2776-2782 VBN denotes floxed
T3068 2783-2789 JJ denotes mutant
T3066 2790-2796 NN denotes allele
T3052 2797-2804 VBZ denotes removes
T3069 2805-2808 DT denotes the
T3071 2809-2817 JJ denotes resident
T3070 2818-2826 NN denotes sequence
T3072 2827-2830 CC denotes and
T3073 2831-2838 VBZ denotes inserts
T3074 2839-2842 DT denotes the
T3076 2843-2849 JJ denotes mutant
T3075 2850-2854 NN denotes gene
T3077 2855-2856 -LRB- denotes (
T3078 2856-2857 CD denotes 7
T3079 2857-2858 -RRB- denotes )
T3080 2858-2859 . denotes .
T3081 2859-2968 sentence denotes Previously, technical difficulties have prevented RMCE from being applied routinely to generate mutant mice.
T3082 2860-2870 RB denotes Previously
T3084 2870-2872 , denotes ,
T3085 2872-2881 JJ denotes technical
T3086 2882-2894 NNS denotes difficulties
T3087 2895-2899 VBD denotes have
T3083 2900-2909 VBN denotes prevented
T3088 2910-2914 NN denotes RMCE
T3089 2915-2919 IN denotes from
T3090 2920-2925 VBG denotes being
T3091 2926-2933 VBN denotes applied
T3092 2934-2943 RB denotes routinely
T3093 2944-2946 TO denotes to
T3094 2947-2955 VB denotes generate
T3095 2956-2962 JJ denotes mutant
T3096 2963-2967 NNS denotes mice
T3097 2967-2968 . denotes .
T3098 2968-3114 sentence denotes For example, exchanges using cassettes with directly repeated loxP sites were inefficient because excisions dominated the intended exchanges (8).
T3099 2969-2972 IN denotes For
T3101 2973-2980 NN denotes example
T3102 2980-2982 , denotes ,
T3103 2982-2991 NNS denotes exchanges
T3104 2992-2997 VBG denotes using
T3105 2998-3007 NNS denotes cassettes
T3106 3008-3012 IN denotes with
T3107 3013-3021 RB denotes directly
T3108 3022-3030 VBN denotes repeated
T3110 3031-3035 NN denotes loxP
T3109 3036-3041 NNS denotes sites
T3100 3042-3046 VBD denotes were
T3111 3047-3058 JJ denotes inefficient
T3112 3059-3066 IN denotes because
T3114 3067-3076 NNS denotes excisions
T3113 3077-3086 VBD denotes dominated
T3115 3087-3090 DT denotes the
T3117 3091-3099 VBN denotes intended
T3116 3100-3109 NNS denotes exchanges
T3118 3110-3111 -LRB- denotes (
T3119 3111-3112 CD denotes 8
T3120 3112-3113 -RRB- denotes )
T3121 3113-3114 . denotes .
T3122 3114-3382 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 3115-3119 NN denotes loxP
T3124 3120-3125 NNS denotes sites
T3126 3126-3130 IN denotes with
T3127 3131-3140 JJ denotes different
T3128 3141-3150 NNS denotes sequences
T3129 3151-3155 VBD denotes were
T3125 3156-3165 VBN denotes generated
T3130 3166-3168 TO denotes to
T3131 3169-3177 VB denotes overcome
T3132 3178-3182 DT denotes this
T3133 3183-3190 NN denotes problem
T3134 3190-3192 , denotes ,
T3135 3192-3195 CC denotes but
T3136 3196-3201 DT denotes these
T3137 3202-3207 NNS denotes sites
T3139 3208-3212 RB denotes also
T3138 3213-3222 VBD denotes underwent
T3140 3223-3237 JJ denotes intramolecular
T3141 3238-3251 NN denotes recombination
T3142 3251-3253 , denotes ,
T3143 3253-3259 VBG denotes making
T3144 3260-3264 NN denotes RMCE
T3145 3265-3274 JJ denotes efficient
T3146 3275-3279 RB denotes only
T3148 3280-3282 IN denotes if
T3149 3283-3286 DT denotes the
T3151 3287-3298 NN denotes replacement
T3150 3299-3307 NN denotes cassette
T3147 3308-3317 VBD denotes contained
T3152 3318-3319 DT denotes a
T3153 3320-3326 NN denotes marker
T3154 3327-3335 VBG denotes enabling
T3155 3336-3345 NN denotes selection
T3156 3346-3348 IN denotes of
T3157 3349-3352 DT denotes the
T3159 3353-3360 VBN denotes desired
T3158 3361-3372 JJ denotes recombinant
T3160 3373-3374 -LRB- denotes (
T3161 3374-3375 CD denotes 7
T3162 3375-3376 , denotes ,
T3163 3376-3377 CD denotes 9
T3164 3377-3378 SYM denotes –
T3165 3378-3380 CD denotes 12
T3166 3380-3381 -RRB- denotes )
T3167 3381-3382 . denotes .
T3168 3382-3538 sentence denotes However, interference resulting from expression of the selection marker and the endogenous gene (13) necessitates strategies to remove the selectable gene.
T3169 3383-3390 RB denotes However
T3171 3390-3392 , denotes ,
T3172 3392-3404 NN denotes interference
T3173 3405-3414 VBG denotes resulting
T3174 3415-3419 IN denotes from
T3175 3420-3430 NN denotes expression
T3176 3431-3433 IN denotes of
T3177 3434-3437 DT denotes the
T3179 3438-3447 NN denotes selection
T3178 3448-3454 NN denotes marker
T3180 3455-3458 CC denotes and
T3181 3459-3462 DT denotes the
T3183 3463-3473 JJ denotes endogenous
T3182 3474-3478 NN denotes gene
T3184 3479-3480 -LRB- denotes (
T3185 3480-3482 CD denotes 13
T3186 3482-3483 -RRB- denotes )
T3170 3484-3496 VBZ denotes necessitates
T3187 3497-3507 NNS denotes strategies
T3188 3508-3510 TO denotes to
T3189 3511-3517 VB denotes remove
T3190 3518-3521 DT denotes the
T3192 3522-3532 JJ denotes selectable
T3191 3533-3537 NN denotes gene
T3193 3537-3538 . denotes .
T3194 3538-3882 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 3539-3547 RB denotes Together
T3197 3547-3549 , denotes ,
T3198 3549-3557 JJ denotes previous
T3199 3558-3565 NNS denotes studies
T3196 3566-3574 VBP denotes indicate
T3200 3575-3579 IN denotes that
T3202 3580-3582 DT denotes an
T3204 3583-3590 JJ denotes optimal
T3203 3591-3595 NN denotes RMCE
T3201 3596-3604 VBZ denotes requires
T3205 3605-3606 -LRB- denotes (
T3206 3606-3607 LS denotes i
T3208 3607-3608 -RRB- denotes )
T3209 3609-3617 VBN denotes inverted
T3210 3618-3630 JJ denotes heterologous
T3211 3631-3635 NN denotes loxP
T3207 3636-3641 NNS denotes sites
T3212 3642-3651 VBG denotes diverging
T3213 3652-3654 IN denotes by
T3214 3655-3657 RB denotes at
T3216 3658-3663 RBS denotes least
T3215 3664-3665 CD denotes 2
T3217 3666-3668 NN denotes nt
T3218 3669-3671 TO denotes to
T3219 3672-3680 VB denotes maximize
T3220 3681-3684 DT denotes the
T3221 3685-3695 NN denotes efficiency
T3222 3696-3698 IN denotes of
T3223 3699-3707 NN denotes exchange
T3224 3708-3711 CC denotes and
T3225 3712-3713 -LRB- denotes (
T3226 3713-3715 LS denotes ii
T3228 3715-3716 -RRB- denotes )
T3229 3717-3719 DT denotes an
T3230 3720-3730 NN denotes expression
T3227 3731-3739 NN denotes cassette
T3231 3740-3748 VBG denotes enabling
T3232 3749-3753 CC denotes both
T3234 3754-3762 JJ denotes positive
T3233 3763-3772 NN denotes selection
T3235 3773-3775 TO denotes to
T3236 3776-3784 VB denotes identify
T3237 3785-3788 DT denotes the
T3239 3789-3796 JJ denotes initial
T3238 3797-3808 JJ denotes recombinant
T3240 3809-3812 CC denotes and
T3241 3813-3817 RB denotes then
T3243 3818-3826 JJ denotes negative
T3242 3827-3836 NN denotes selection
T3244 3837-3839 TO denotes to
T3245 3840-3846 VB denotes obtain
T3246 3847-3848 DT denotes a
T3248 3849-3850 `` denotes ‘
T3250 3850-3856 NN denotes marker
T3251 3856-3857 HYPH denotes -
T3249 3857-3861 JJ denotes free
T3252 3861-3862 '' denotes ’
T3253 3863-3869 JJ denotes mutant
T3247 3870-3876 NN denotes allele
T3254 3877-3878 -LRB- denotes (
T3255 3878-3880 CD denotes 14
T3256 3880-3881 -RRB- denotes )
T3257 3881-3882 . denotes .
T3258 3882-3963 sentence denotes Most RMCE experiments have been performed at random sites in somatic cell lines.
T3259 3883-3887 JJS denotes Most
T3261 3888-3892 NN denotes RMCE
T3260 3893-3904 NNS denotes experiments
T3263 3905-3909 VBP denotes have
T3264 3910-3914 VBN denotes been
T3262 3915-3924 VBN denotes performed
T3265 3925-3927 IN denotes at
T3266 3928-3934 JJ denotes random
T3267 3935-3940 NNS denotes sites
T3268 3941-3943 IN denotes in
T3269 3944-3951 JJ denotes somatic
T3270 3952-3956 NN denotes cell
T3271 3957-3962 NNS denotes lines
T3272 3962-3963 . denotes .
T3273 3963-4173 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 3964-3968 RB denotes Only
T3276 3969-3970 DT denotes a
T3277 3971-3974 JJ denotes few
T3278 3975-3981 JJ denotes mutant
T3275 3982-3986 NNS denotes mice
T3280 3987-3996 VBN denotes generated
T3281 3997-3999 IN denotes by
T3282 4000-4004 NN denotes RMCE
T3283 4005-4007 IN denotes in
T3284 4008-4010 NN denotes ES
T3285 4011-4016 NNS denotes cells
T3286 4017-4021 VBP denotes have
T3287 4022-4026 VBN denotes been
T3279 4027-4035 VBN denotes reported
T3288 4035-4037 , denotes ,
T3289 4037-4040 CC denotes but
T3290 4041-4044 DT denotes the
T3291 4045-4049 NN denotes RMCE
T3293 4050-4064 RB denotes systematically
T3292 4065-4075 VBD denotes introduced
T3294 4076-4077 DT denotes a
T3296 4078-4088 JJ denotes selectable
T3295 4089-4095 NN denotes marker
T3297 4096-4097 -LRB- denotes (
T3298 4097-4102 CD denotes 15–17
T3299 4102-4103 -RRB- denotes )
T3300 4103-4105 , denotes ,
T3301 4105-4107 CC denotes or
T3302 4107-4109 , denotes ,
T3303 4109-4113 WRB denotes when
T3304 4114-4120 VBN denotes tested
T3306 4121-4128 IN denotes without
T3307 4129-4131 DT denotes an
T3309 4132-4140 JJ denotes incoming
T3308 4141-4147 NN denotes marker
T3310 4147-4149 , denotes ,
T3305 4149-4155 VBD denotes proved
T3311 4156-4167 JJ denotes inefficient
T3312 4168-4169 -LRB- denotes (
T3313 4169-4171 CD denotes 12
T3314 4171-4172 -RRB- denotes )
T3315 4172-4173 . denotes .
T3316 4173-4221 sentence denotes A recent report disclosed an additional problem
T3317 4174-4175 DT denotes A
T3319 4176-4182 JJ denotes recent
T3318 4183-4189 NN denotes report
T3320 4190-4199 VBD denotes disclosed
T3321 4200-4202 DT denotes an
T3323 4203-4213 JJ denotes additional
T3322 4214-4221 NN denotes problem
T3324 4221-4223 : denotes :
T3326 4221-4412 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 4223-4226 DT denotes the
T3329 4227-4237 NN denotes Hygromycin
T3331 4237-4238 HYPH denotes –
T3330 4238-4247 NN denotes Thymidine
T3332 4248-4254 NN denotes Kinase
T3333 4255-4261 NN denotes fusion
T3328 4262-4266 NN denotes gene
T3334 4267-4271 VBN denotes used
T3335 4272-4276 RBS denotes most
T3336 4277-4287 RB denotes frequently
T3337 4288-4291 IN denotes for
T3338 4292-4300 JJ denotes positive
T3340 4300-4301 HYPH denotes /
T3339 4301-4309 JJ denotes negative
T3341 4310-4319 NN denotes selection
T3342 4320-4322 IN denotes in
T3343 4323-4327 NN denotes RMCE
T3344 4327-4329 , denotes ,
T3325 4329-4334 VBZ denotes leads
T3345 4335-4337 IN denotes to
T3346 4338-4343 NN denotes mouse
T3347 4344-4353 NN denotes sterility
T3348 4353-4355 , denotes ,
T3349 4355-4357 IN denotes so
T3351 4358-4362 IN denotes that
T3352 4363-4372 NNS denotes exchanges
T3353 4373-4376 MD denotes can
T3354 4377-4381 RB denotes only
T3355 4382-4384 VB denotes be
T3350 4385-4394 VBN denotes performed
T3356 4395-4397 IN denotes in
T3357 4398-4400 NN denotes ES
T3358 4401-4406 NNS denotes cells
T3359 4407-4408 -LRB- denotes (
T3360 4408-4410 CD denotes 16
T3361 4410-4411 -RRB- denotes )
T3362 4411-4412 . denotes .
T3363 4412-4663 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 4413-4416 DT denotes The
T3366 4417-4421 NN denotes RMCE
T3365 4422-4430 NN denotes strategy
T3368 4431-4440 VBN denotes presented
T3369 4441-4445 RB denotes here
T3367 4446-4452 VBZ denotes relies
T3370 4453-4455 IN denotes on
T3371 4456-4459 DT denotes the
T3373 4460-4470 VBN denotes integrated
T3372 4471-4474 NN denotes use
T3374 4475-4477 IN denotes of
T3375 4478-4486 VBN denotes inverted
T3377 4487-4499 JJ denotes heterologous
T3378 4500-4504 NN denotes loxP
T3376 4505-4510 NNS denotes sites
T3379 4510-4512 , denotes ,
T3380 4512-4513 DT denotes a
T3382 4514-4522 JJ denotes positive
T3384 4522-4523 HYPH denotes /
T3383 4523-4531 JJ denotes negative
T3385 4532-4541 NN denotes selection
T3381 4542-4548 NN denotes marker
T3386 4549-4553 WDT denotes that
T3387 4554-4563 VBZ denotes preserves
T3388 4564-4567 DT denotes the
T3390 4568-4576 NN denotes germline
T3389 4577-4585 NN denotes capacity
T3391 4586-4588 IN denotes of
T3392 4589-4591 NN denotes ES
T3393 4592-4597 NNS denotes cells
T3394 4597-4599 , denotes ,
T3395 4599-4602 CC denotes and
T3396 4603-4606 DT denotes the
T3397 4607-4612 NN denotes power
T3398 4613-4615 IN denotes of
T3399 4616-4621 NN denotes mouse
T3400 4622-4630 NN denotes genetics
T3401 4631-4633 TO denotes to
T3402 4634-4642 VB denotes expedite
T3403 4643-4653 JJ denotes phenotypic
T3404 4654-4662 NNS denotes analyses
T3405 4662-4663 . denotes .
T3406 4663-4897 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 4664-4666 PRP denotes We
T3408 4667-4671 VBP denotes show
T3409 4672-4676 IN denotes that
T3411 4677-4680 PRP$ denotes our
T3412 4681-4689 NN denotes approach
T3410 4690-4697 VBZ denotes enables
T3413 4698-4707 JJ denotes efficient
T3414 4708-4717 NN denotes targeting
T3415 4718-4720 IN denotes of
T3416 4721-4727 JJ denotes marker
T3418 4727-4728 HYPH denotes -
T3417 4728-4732 JJ denotes free
T3419 4733-4742 NNS denotes mutations
T3420 4743-4745 IN denotes at
T3421 4746-4749 DT denotes the
T3423 4750-4753 NN denotes p53
T3422 4754-4759 NN denotes locus
T3424 4760-4762 IN denotes in
T3425 4763-4765 NN denotes ES
T3426 4766-4771 NNS denotes cells
T3427 4772-4774 TO denotes to
T3428 4775-4783 VB denotes generate
T3429 4784-4790 JJ denotes mutant
T3430 4791-4795 NNS denotes mice
T3431 4795-4797 , denotes ,
T3432 4797-4800 CC denotes but
T3433 4801-4805 RBR denotes more
T3434 4806-4817 RB denotes importantly
T3436 4817-4819 , denotes ,
T3437 4819-4821 PRP denotes it
T3438 4822-4824 VBZ denotes is
T3435 4825-4832 VBN denotes Adapted
T3439 4833-4836 IN denotes for
T3440 4837-4846 VBG denotes targeting
T3441 4847-4849 IN denotes in
T3442 4850-4857 JJ denotes Somatic
T3443 4858-4863 NNS denotes cells
T3444 4864-4866 TO denotes to
T3445 4867-4877 VB denotes Accelerate
T3446 4878-4889 NN denotes Phenotyping
T3447 4890-4891 -LRB- denotes (
T3448 4891-4895 NN denotes ASAP
T3449 4895-4896 -RRB- denotes )
T3450 4896-4897 . denotes .
T3451 4897-4996 sentence denotes Because it relies on very general principles, RMCE-ASAP could be applied to any locus of interest.
T3452 4898-4905 IN denotes Because
T3454 4906-4908 PRP denotes it
T3453 4909-4915 VBZ denotes relies
T3456 4916-4918 IN denotes on
T3457 4919-4923 RB denotes very
T3459 4924-4931 JJ denotes general
T3458 4932-4942 NNS denotes principles
T3460 4942-4944 , denotes ,
T3461 4944-4948 NN denotes RMCE
T3463 4948-4949 HYPH denotes -
T3462 4949-4953 NN denotes ASAP
T3464 4954-4959 MD denotes could
T3465 4960-4962 VB denotes be
T3455 4963-4970 VBN denotes applied
T3466 4971-4973 IN denotes to
T3467 4974-4977 DT denotes any
T3468 4978-4983 NN denotes locus
T3469 4984-4986 IN denotes of
T3470 4987-4995 NN denotes interest
T3471 4995-4996 . denotes .
T4611 5021-5030 NN denotes Targeting
T4612 5031-5040 NN denotes construct
T4613 5041-5044 IN denotes for
T4614 5045-5046 DT denotes a
T4616 5047-5050 NN denotes p53
T4617 5051-5055 NN denotes RMCE
T4619 5055-5056 HYPH denotes -
T4618 5056-5061 JJ denotes ready
T4615 5062-5067 NN denotes locus
T4620 5067-5181 sentence denotes Details for plasmid construction are available upon request owing to space limitations mandating a brief outline.
T4621 5068-5075 NNS denotes Details
T4623 5076-5079 IN denotes for
T4624 5080-5087 NN denotes plasmid
T4625 5088-5100 NN denotes construction
T4622 5101-5104 VBP denotes are
T4626 5105-5114 JJ denotes available
T4627 5115-5119 IN denotes upon
T4628 5120-5127 NN denotes request
T4629 5128-5133 VBG denotes owing
T4630 5134-5136 IN denotes to
T4631 5137-5142 NN denotes space
T4632 5143-5154 NNS denotes limitations
T4633 5155-5164 VBG denotes mandating
T4634 5165-5166 DT denotes a
T4636 5167-5172 JJ denotes brief
T4635 5173-5180 NN denotes outline
T4637 5180-5181 . denotes .
T4638 5181-5327 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 5182-5184 PRP denotes We
T4640 5185-5192 VBD denotes started
T4641 5193-5197 IN denotes from
T4642 5198-5199 DT denotes a
T4644 5200-5207 NN denotes plasmid
T4645 5208-5210 NN denotes L3
T4646 5210-5211 HYPH denotes -
T4643 5211-5213 NN denotes 1L
T4647 5214-5224 VBG denotes containing
T4648 5225-5237 JJ denotes heterologous
T4650 5238-5242 NN denotes loxP
T4649 5243-5248 NNS denotes sites
T4651 5249-5250 -LRB- denotes (
T4653 5250-5252 NN denotes L3
T4654 5253-5255 VBZ denotes is
T4655 5256-5259 DT denotes the
T4657 5260-5266 JJ denotes mutant
T4656 5267-5274 NN denotes loxP257
T4658 5275-5283 RB denotes recently
T4659 5284-5293 VBN denotes described
T4660 5294-5295 -LRB- denotes (
T4661 5295-5297 CD denotes 14
T4662 5297-5298 -RRB- denotes )
T4663 5298-5300 , denotes ,
T4664 5300-5302 NN denotes 1L
T4652 5303-5305 VBZ denotes is
T4665 5306-5308 DT denotes an
T4667 5309-5317 VBN denotes inverted
T4668 5318-5320 NN denotes WT
T4666 5321-5325 NN denotes loxP
T4669 5325-5326 -RRB- denotes )
T4670 5326-5327 . denotes .
T4671 5327-5467 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 5328-5331 DT denotes The
T4674 5332-5334 NN denotes WT
T4673 5335-5339 NN denotes loxP
T4676 5340-5343 CC denotes and
T4677 5344-5351 NN denotes loxP257
T4675 5352-5358 VBP denotes differ
T4679 5359-5361 IN denotes in
T4680 5362-5367 PRP$ denotes their
T4682 5368-5374 NN denotes spacer
T4681 5375-5384 NNS denotes sequences
T4683 5384-5386 : denotes :
T4684 5386-5389 DT denotes the
T4686 5390-5396 NN denotes spacer
T4685 5397-5405 NN denotes sequence
T4678 5406-5408 VBZ denotes is
T4687 5409-5410 CD denotes 5
T4688 5410-5411 SYM denotes ′
T4689 5411-5412 HYPH denotes -
T4690 5412-5420 JJ denotes ATGTATGC
T4691 5420-5421 HYPH denotes -
T4692 5421-5422 CD denotes 3
T4693 5422-5423 SYM denotes ′
T4694 5424-5427 IN denotes for
T4695 5428-5430 NN denotes WT
T4696 5431-5435 NN denotes loxP
T4697 5436-5439 CC denotes and
T4698 5440-5441 CD denotes 5
T4700 5441-5442 SYM denotes ′
T4701 5442-5443 HYPH denotes -
T4699 5443-5451 NN denotes AAGTCTCC
T4702 5451-5452 HYPH denotes -
T4703 5452-5453 CD denotes 3
T4704 5453-5454 SYM denotes ′
T4705 5455-5458 IN denotes for
T4706 5459-5466 NN denotes loxP257
T4707 5466-5467 . denotes .
T4708 5467-5771 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 5468-5471 DT denotes The
T4711 5472-5477 CD denotes three
T4710 5478-5487 NNS denotes mutations
T4713 5488-5490 IN denotes in
T4714 5491-5494 DT denotes the
T4716 5495-5502 NN denotes loxP257
T4717 5503-5509 NN denotes spacer
T4715 5510-5518 NN denotes sequence
T4712 5519-5526 VBP denotes prevent
T4719 5527-5529 PRP denotes it
T4721 5530-5532 TO denotes to
T4720 5533-5542 VB denotes recombine
T4722 5543-5547 IN denotes with
T4723 5548-5550 NN denotes WT
T4724 5551-5555 NN denotes loxP
T4725 5555-5557 , denotes ,
T4726 5557-5565 VBG denotes ensuring
T4727 5566-5575 JJ denotes efficient
T4728 5576-5580 NN denotes RMCE
T4729 5581-5583 IN denotes in
T4730 5584-5591 JJ denotes several
T4732 5592-5596 NN denotes cell
T4731 5597-5602 NNS denotes lines
T4733 5602-5604 : denotes :
T4734 5604-5612 JJ denotes accurate
T4735 5613-5617 NN denotes RMCE
T4736 5618-5622 IN denotes with
T4737 5623-5628 DT denotes these
T4739 5629-5633 NN denotes loxP
T4738 5634-5639 NNS denotes sites
T4718 5640-5648 VBD denotes occurred
T4740 5649-5653 IN denotes with
T4741 5654-5656 DT denotes an
T4743 5657-5664 JJ denotes average
T4742 5665-5674 NN denotes frequency
T4744 5675-5677 IN denotes of
T4745 5678-5680 CD denotes 81
T4746 5680-5681 NN denotes %
T4747 5682-5684 IN denotes at
T4748 5685-5688 CD denotes two
T4749 5689-5693 NNS denotes loci
T4750 5694-5696 IN denotes in
T4751 5697-5700 NN denotes CHO
T4752 5701-5706 NNS denotes cells
T4753 5707-5710 CC denotes and
T4754 5711-5713 DT denotes an
T4756 5714-5721 JJ denotes average
T4755 5722-5731 NN denotes frequency
T4757 5732-5734 IN denotes of
T4758 5735-5737 CD denotes 69
T4759 5737-5738 NN denotes %
T4760 5739-5741 IN denotes at
T4761 5742-5746 CD denotes four
T4762 5747-5751 NNS denotes loci
T4763 5752-5754 IN denotes in
T4764 5755-5759 NN denotes Hela
T4765 5760-5765 NNS denotes cells
T4766 5766-5767 -LRB- denotes (
T4767 5767-5769 CD denotes 14
T4768 5769-5770 -RRB- denotes )
T4769 5770-5771 . denotes .
T4770 5771-5895 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 5772-5775 DT denotes The
T4773 5776-5778 NN denotes L3
T4775 5778-5779 HYPH denotes -
T4774 5779-5781 NN denotes 1L
T4772 5782-5789 NN denotes plasmid
T4777 5790-5793 VBD denotes was
T4778 5794-5799 RB denotes first
T4776 5800-5808 VBN denotes modified
T4779 5809-5811 TO denotes to
T4780 5812-5819 VB denotes include
T4781 5820-5821 DT denotes a
T4782 5822-5826 NN denotes ClaI
T4783 5827-5830 CC denotes and
T4784 5831-5832 DT denotes a
T4786 5833-5837 NN denotes FseI
T4785 5838-5842 NN denotes site
T4787 5843-5850 IN denotes between
T4788 5851-5854 DT denotes the
T4790 5855-5859 NN denotes LoxP
T4789 5860-5865 NNS denotes sites
T4791 5865-5867 , denotes ,
T4792 5867-5874 VBG denotes leading
T4793 5875-5877 IN denotes to
T4794 5878-5885 NN denotes plasmid
T4796 5886-5888 NN denotes L3
T4797 5888-5889 HYPH denotes -
T4798 5889-5891 NN denotes CF
T4799 5891-5892 HYPH denotes -
T4795 5892-5894 NN denotes 1L
T4800 5894-5895 . denotes .
T4801 5895-6157 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 5896-5898 PRP denotes We
T4804 5899-5903 RB denotes next
T4803 5904-5912 VBD denotes modified
T4805 5913-5914 DT denotes a
T4807 5915-5922 NN denotes puroΔTK
T4806 5923-5930 NN denotes plasmid
T4808 5931-5932 -LRB- denotes (
T4809 5932-5937 NN denotes YTC37
T4810 5937-5939 , denotes ,
T4811 5939-5940 DT denotes a
T4813 5941-5945 NN denotes kind
T4812 5946-5950 NN denotes gift
T4814 5951-5955 IN denotes from
T4815 5956-5958 NNP denotes A.
T4816 5959-5966 NNP denotes Bradley
T4817 5966-5967 -RRB- denotes )
T4818 5968-5970 IN denotes by
T4819 5971-5976 VBG denotes using
T4820 5977-5993 NNS denotes oligonucleotides
T4821 5994-5996 TO denotes to
T4822 5997-6004 VB denotes destroy
T4823 6005-6006 DT denotes a
T4825 6007-6011 NN denotes NotI
T4824 6012-6016 NN denotes site
T4826 6017-6027 RB denotes downstream
T4827 6028-6030 IN denotes of
T4828 6031-6034 DT denotes the
T4830 6035-6042 NN denotes puroΔTK
T4829 6043-6047 NN denotes gene
T4831 6048-6051 CC denotes and
T4832 6052-6061 VB denotes introduce
T4833 6062-6063 DT denotes a
T4835 6064-6068 NN denotes NotI
T4834 6069-6073 NN denotes site
T4836 6074-6082 RB denotes upstream
T4837 6082-6084 , denotes ,
T4838 6084-6087 CC denotes and
T4839 6088-6089 DT denotes a
T4841 6090-6094 NN denotes FseI
T4840 6095-6099 NN denotes site
T4842 6100-6110 RB denotes downstream
T4843 6111-6113 IN denotes of
T4844 6114-6117 DT denotes the
T4845 6118-6122 NN denotes gene
T4846 6123-6124 -LRB- denotes (
T4847 6124-6131 VBG denotes leading
T4848 6132-6134 IN denotes to
T4849 6135-6142 NN denotes plasmid
T4851 6143-6145 NN denotes CN
T4852 6145-6146 HYPH denotes -
T4853 6146-6153 NN denotes PuroΔTK
T4854 6153-6154 HYPH denotes -
T4850 6154-6155 NN denotes F
T4855 6155-6156 -RRB- denotes )
T4856 6156-6157 . denotes .
T4857 6157-6405 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 6158-6162 RB denotes Next
T4860 6162-6164 , denotes ,
T4861 6164-6165 DT denotes a
T4863 6166-6170 NN denotes PmlI
T4865 6170-6171 HYPH denotes -
T4864 6171-6175 NN denotes MfeI
T4866 6176-6179 CD denotes 6.3
T4867 6180-6182 NN denotes kb
T4862 6183-6191 NN denotes fragment
T4868 6192-6196 IN denotes from
T4869 6197-6202 NN denotes Trp53
T4870 6203-6206 VBD denotes was
T4859 6207-6216 VBN denotes subcloned
T4871 6217-6219 IN denotes in
T4872 6220-6221 DT denotes a
T4874 6222-6230 VBN denotes modified
T4875 6231-6242 NN denotes pBluescript
T4873 6243-6248 NN denotes KSII+
T4876 6249-6250 -LRB- denotes (
T4877 6250-6253 NN denotes pBS
T4878 6253-6255 , denotes ,
T4879 6255-6265 NNP denotes Stratagene
T4880 6265-6266 -RRB- denotes )
T4881 6266-6268 , denotes ,
T4882 6268-6271 CC denotes and
T4883 6272-6275 DT denotes the
T4885 6276-6285 VBG denotes resulting
T4884 6286-6293 NN denotes plasmid
T4887 6294-6297 VBD denotes was
T4886 6298-6306 VBN denotes digested
T4888 6307-6311 IN denotes with
T4889 6312-6316 NN denotes SwaI
T4890 6317-6319 TO denotes to
T4891 6320-6329 VB denotes introduce
T4892 6330-6332 DT denotes an
T4894 6333-6337 NN denotes EagI
T4893 6338-6342 NN denotes site
T4895 6342-6344 , denotes ,
T4896 6344-6351 VBG denotes leading
T4897 6352-6354 IN denotes to
T4898 6355-6364 NN denotes p53PmlEag
T4899 6364-6366 , denotes ,
T4900 6366-6367 DT denotes a
T4901 6368-6375 NN denotes plasmid
T4902 6376-6386 VBG denotes containing
T4903 6387-6392 NNS denotes exons
T4905 6393-6394 CD denotes 2
T4906 6394-6395 HYPH denotes –
T4904 6395-6397 CD denotes 11
T4907 6398-6400 IN denotes of
T4908 6401-6404 NN denotes p53
T4909 6404-6405 . denotes .
T4910 6405-6650 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 6406-6408 PRP denotes We
T4913 6409-6413 RB denotes then
T4912 6414-6422 VBD denotes inserted
T4914 6423-6424 DT denotes a
T4916 6425-6428 CD denotes 5.5
T4917 6429-6431 NN denotes kb
T4918 6432-6436 NN denotes ClaI
T4920 6436-6437 HYPH denotes -
T4919 6437-6441 NN denotes EagI
T4915 6442-6450 NN denotes fragment
T4921 6451-6455 IN denotes from
T4922 6456-6465 NN denotes p53PmlEag
T4923 6466-6468 IN denotes in
T4924 6469-6476 NN denotes plasmid
T4926 6477-6479 NN denotes CN
T4927 6479-6480 HYPH denotes -
T4928 6480-6487 NN denotes PuroΔTK
T4929 6487-6488 HYPH denotes -
T4925 6488-6489 NN denotes F
T4930 6490-6498 VBN denotes digested
T4931 6499-6501 IN denotes by
T4932 6502-6506 NN denotes ClaI
T4933 6507-6510 CC denotes and
T4934 6511-6515 NN denotes NotI
T4935 6515-6517 , denotes ,
T4936 6517-6520 CC denotes and
T4937 6521-6529 VBD denotes inserted
T4938 6530-6533 DT denotes the
T4940 6534-6543 VBG denotes resulting
T4939 6544-6552 NN denotes fragment
T4941 6553-6560 IN denotes between
T4942 6561-6564 DT denotes the
T4944 6565-6569 NN denotes loxP
T4943 6570-6575 NNS denotes sites
T4945 6576-6578 IN denotes of
T4946 6579-6581 NN denotes L3
T4948 6581-6582 HYPH denotes -
T4949 6582-6584 NN denotes CF
T4950 6584-6585 HYPH denotes -
T4947 6585-6587 NN denotes 1L
T4951 6588-6590 IN denotes by
T4952 6591-6595 NN denotes ClaI
T4954 6596-6599 CC denotes and
T4955 6600-6604 NN denotes FseI
T4953 6605-6614 NN denotes digestion
T4956 6614-6616 , denotes ,
T4957 6616-6623 VBG denotes leading
T4958 6624-6626 IN denotes to
T4959 6627-6629 NN denotes L3
T4961 6629-6630 HYPH denotes -
T4962 6630-6646 NN denotes p53PmlEagPuroΔTK
T4963 6646-6647 HYPH denotes -
T4960 6647-6649 NN denotes 1L
T4964 6649-6650 . denotes .
T4965 6650-7240 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 6651-6653 PRP denotes We
T4968 6654-6658 RB denotes next
T4967 6659-6669 VBD denotes engineered
T4970 6670-6671 DT denotes a
T4971 6672-6679 NN denotes plasmid
T4972 6680-6690 VBG denotes containing
T4973 6691-6694 DT denotes the
T4974 6695-6701 NN denotes region
T4975 6702-6705 IN denotes for
T4976 6706-6707 CD denotes 3
T4977 6707-6708 SYM denotes ′
T4978 6708-6709 SYM denotes -
T4979 6709-6717 JJ denotes homology
T4980 6718-6728 RB denotes downstream
T4981 6729-6731 IN denotes of
T4982 6732-6735 DT denotes the
T4984 6736-6739 NN denotes p53
T4983 6740-6744 NN denotes gene
T4985 6745-6748 CC denotes and
T4986 6749-6752 DT denotes the
T4988 6753-6756 NN denotes DTA
T4987 6757-6761 NN denotes gene
T4989 6762-6764 IN denotes in
T4990 6765-6768 CD denotes two
T4991 6769-6774 NNS denotes steps
T4992 6774-6776 : denotes :
T4993 6776-6777 -LRB- denotes (
T4994 6777-6778 LS denotes i
T4995 6778-6779 -RRB- denotes )
T4996 6780-6782 PRP denotes we
T4969 6783-6792 VBD denotes performed
T4997 6793-6794 DT denotes a
T4999 6795-6800 CD denotes three
T5001 6800-6801 HYPH denotes -
T5000 6801-6804 NN denotes way
T4998 6805-6813 NN denotes ligation
T5002 6814-6821 IN denotes between
T5003 6822-6823 DT denotes a
T5005 6824-6832 VBN denotes modified
T5004 6833-6836 NN denotes pBS
T5006 6837-6845 VBN denotes digested
T5007 6846-6848 IN denotes by
T5008 6849-6856 NN denotes HindIII
T5009 6857-6860 CC denotes and
T5010 6861-6865 NN denotes NotI
T5011 6865-6867 , denotes ,
T5012 6867-6868 DT denotes a
T5014 6869-6876 NN denotes HindIII
T5016 6876-6877 HYPH denotes -
T5015 6877-6882 NN denotes EcoRI
T5013 6883-6891 NN denotes fragment
T5017 6892-6896 IN denotes from
T5018 6897-6902 NN denotes Trp53
T5019 6903-6906 IN denotes for
T5020 6907-6908 CD denotes 3
T5022 6908-6909 SYM denotes ′
T5021 6909-6917 NN denotes homology
T5023 6918-6921 CC denotes and
T5024 6922-6924 DT denotes an
T5026 6925-6930 NN denotes EcoRI
T5028 6930-6931 HYPH denotes -
T5027 6931-6935 NN denotes NotI
T5025 6936-6944 NN denotes fragment
T5029 6945-6955 VBG denotes containing
T5030 6956-6959 DT denotes the
T5032 6960-6963 NN denotes DTA
T5031 6964-6968 NN denotes gene
T5033 6968-6970 , denotes ,
T5034 6970-6974 IN denotes from
T5035 6975-6982 NN denotes plasmid
T5036 6983-6992 NN denotes pgkdtabpa
T5037 6993-6994 -LRB- denotes (
T5038 6994-6998 JJ denotes kind
T5039 6999-7003 NN denotes gift
T5040 7004-7006 IN denotes of
T5041 7007-7009 NNP denotes P.
T5042 7010-7017 NNP denotes Soriano
T5043 7017-7018 -RRB- denotes )
T5044 7018-7020 , denotes ,
T5045 7020-7027 VBG denotes leading
T5046 7028-7030 IN denotes to
T5047 7031-7038 NN denotes plasmid
T5049 7039-7040 CD denotes 3
T5050 7040-7041 SYM denotes ′
T5051 7042-7043 SYM denotes +
T5048 7044-7047 NN denotes DTA
T5052 7048-7051 CC denotes and
T5053 7052-7053 -LRB- denotes (
T5054 7053-7055 LS denotes ii
T5056 7055-7056 -RRB- denotes )
T5057 7057-7064 IN denotes because
T5059 7065-7068 DT denotes the
T5061 7069-7075 NN denotes Bsu36I
T5063 7075-7076 HYPH denotes -
T5062 7076-7081 NN denotes EcoRI
T5060 7082-7088 NN denotes region
T5064 7089-7099 RB denotes downstream
T5065 7100-7102 IN denotes of
T5066 7103-7106 NN denotes p53
T5058 7107-7115 VBZ denotes contains
T5067 7116-7126 JJ denotes repetitive
T5068 7127-7136 NNS denotes sequences
T5069 7137-7138 -LRB- denotes (
T5070 7138-7140 NNP denotes F.
T5071 7141-7147 NNP denotes Toledo
T5072 7148-7151 CC denotes and
T5073 7152-7154 NNP denotes G.
T5074 7155-7157 NNP denotes M.
T5075 7158-7162 NNP denotes Wahl
T5076 7162-7164 , denotes ,
T5077 7164-7175 JJ denotes unpublished
T5078 7176-7180 NNS denotes data
T5079 7180-7181 -RRB- denotes )
T5080 7181-7183 , denotes ,
T5081 7183-7185 PRP denotes we
T5082 7186-7191 RB denotes later
T5055 7192-7199 VBD denotes deleted
T5083 7200-7204 DT denotes this
T5084 7205-7211 NN denotes region
T5085 7211-7213 , denotes ,
T5086 7213-7215 TO denotes to
T5087 7216-7222 VB denotes obtain
T5088 7223-7230 NN denotes plasmid
T5090 7231-7232 CD denotes 3
T5091 7232-7233 SYM denotes ′
T5092 7234-7235 SYM denotes +
T5089 7236-7239 NN denotes DTA
T5093 7239-7240 . denotes .
T5094 7240-7363 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 7241-7244 DT denotes The
T5097 7245-7246 CD denotes 5
T5098 7246-7247 SYM denotes ′
T5096 7248-7256 NN denotes homology
T5099 7257-7265 VBZ denotes consists
T5100 7266-7268 IN denotes of
T5101 7269-7270 DT denotes a
T5103 7271-7274 CD denotes 3.4
T5104 7275-7277 NN denotes kb
T5106 7277-7278 HYPH denotes -
T5105 7278-7282 JJ denotes long
T5107 7283-7288 NN denotes BamHI
T5109 7288-7289 HYPH denotes -
T5108 7289-7293 NN denotes PmlI
T5102 7294-7302 NN denotes fragment
T5110 7303-7307 IN denotes from
T5111 7308-7314 NN denotes intron
T5112 7315-7316 CD denotes 1
T5113 7317-7319 IN denotes of
T5114 7320-7323 NN denotes p53
T5115 7324-7330 VBN denotes cloned
T5116 7331-7333 IN denotes in
T5117 7334-7335 DT denotes a
T5119 7336-7344 VBN denotes modified
T5118 7345-7348 NN denotes pBS
T5120 7349-7350 -LRB- denotes (
T5121 7350-7357 NN denotes plasmid
T5122 7358-7360 NN denotes p5
T5123 7360-7361 SYM denotes ′
T5124 7361-7362 -RRB- denotes )
T5125 7362-7363 . denotes .
T5126 7363-7504 sentence denotes Finally, appropriate fragments from plasmids p5′, L3-p53PmlEagPuroΔTK-1L, and 3′ + DTA were assembled in a modified pSP72 plasmid (Promega).
T5127 7364-7371 RB denotes Finally
T5129 7371-7373 , denotes ,
T5130 7373-7384 JJ denotes appropriate
T5131 7385-7394 NNS denotes fragments
T5132 7395-7399 IN denotes from
T5133 7400-7408 NNS denotes plasmids
T5134 7409-7411 NN denotes p5
T5135 7411-7412 SYM denotes ′
T5136 7412-7414 , denotes ,
T5137 7414-7416 NN denotes L3
T5139 7416-7417 HYPH denotes -
T5140 7417-7433 NN denotes p53PmlEagPuroΔTK
T5141 7433-7434 HYPH denotes -
T5138 7434-7436 NN denotes 1L
T5142 7436-7438 , denotes ,
T5143 7438-7441 CC denotes and
T5144 7442-7443 CD denotes 3
T5146 7443-7444 SYM denotes ′
T5147 7445-7446 SYM denotes +
T5145 7447-7450 NN denotes DTA
T5148 7451-7455 VBD denotes were
T5128 7456-7465 VBN denotes assembled
T5149 7466-7468 IN denotes in
T5150 7469-7470 DT denotes a
T5152 7471-7479 VBN denotes modified
T5153 7480-7485 NN denotes pSP72
T5151 7486-7493 NN denotes plasmid
T5154 7494-7495 -LRB- denotes (
T5155 7495-7502 NNP denotes Promega
T5156 7502-7503 -RRB- denotes )
T5157 7503-7504 . denotes .
T5158 7504-7750 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 7505-7512 NN denotes Plasmid
T5160 7513-7517 NN denotes Flox
T5162 7517-7519 , denotes ,
T5163 7519-7522 DT denotes the
T5165 7523-7532 VBG denotes resulting
T5166 7533-7542 NN denotes targeting
T5164 7543-7552 NN denotes construct
T5167 7552-7554 , denotes ,
T5168 7554-7557 VBD denotes was
T5161 7558-7566 VBN denotes verified
T5169 7567-7569 IN denotes by
T5170 7570-7581 NN denotes restriction
T5171 7582-7590 NN denotes analysis
T5172 7590-7592 , denotes ,
T5173 7592-7596 RB denotes then
T5174 7597-7606 VBN denotes sequenced
T5175 7607-7612 VBG denotes using
T5176 7613-7615 CD denotes 30
T5177 7616-7623 NNS denotes primers
T5178 7624-7630 VBN denotes chosen
T5179 7631-7633 TO denotes to
T5181 7634-7643 RB denotes precisely
T5180 7644-7650 VB denotes verify
T5182 7651-7654 DT denotes all
T5184 7655-7658 NN denotes p53
T5185 7659-7665 NN denotes coding
T5183 7666-7675 NNS denotes sequences
T5186 7675-7677 , denotes ,
T5187 7677-7680 DT denotes all
T5189 7681-7685 NN denotes exon
T5191 7685-7686 HYPH denotes –
T5190 7686-7692 NN denotes intron
T5188 7693-7702 NNS denotes junctions
T5192 7703-7706 CC denotes and
T5193 7707-7710 DT denotes the
T5194 7711-7720 NNS denotes sequences
T5195 7721-7723 IN denotes at
T5196 7724-7727 CC denotes and
T5197 7728-7734 IN denotes around
T5198 7735-7738 DT denotes the
T5200 7739-7743 NN denotes loxP
T5199 7744-7749 NNS denotes sites
T5201 7749-7750 . denotes .
T5752 7752-7760 NN denotes Exchange
T5753 7761-7771 NNS denotes constructs
T5754 7771-7773 : denotes :
T5755 7773-7779 VBG denotes making
T5756 7780-7783 DT denotes the
T5757 7784-7790 NN denotes p53GFP
T5759 7791-7794 CC denotes and
T5760 7795-7798 NN denotes p53
T5762 7798-7799 NN denotes Δ
T5761 7799-7803 NN denotes PGFP
T5758 7804-7812 NNS denotes plasmids
T5763 7812-8036 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 7813-7815 TO denotes To
T5765 7816-7820 VB denotes make
T5767 7821-7822 DT denotes a
T5769 7823-7826 NN denotes p53
T5771 7826-7827 HYPH denotes -
T5770 7827-7830 NN denotes GFP
T5772 7831-7837 NN denotes fusion
T5768 7838-7845 NN denotes protein
T5773 7845-7847 , denotes ,
T5774 7847-7849 PRP denotes we
T5775 7850-7855 RB denotes first
T5766 7856-7865 VBD denotes subcloned
T5776 7866-7867 DT denotes a
T5778 7868-7873 NN denotes SacII
T5780 7873-7874 HYPH denotes -
T5779 7874-7881 NN denotes HindIII
T5777 7882-7890 NN denotes fragment
T5781 7891-7893 IN denotes of
T5782 7894-7897 DT denotes the
T5784 7898-7901 NN denotes p53
T5783 7902-7907 NN denotes locus
T5785 7908-7909 -LRB- denotes (
T5786 7909-7922 VBG denotes corresponding
T5787 7923-7925 IN denotes to
T5788 7926-7930 NN denotes part
T5789 7931-7933 IN denotes of
T5790 7934-7938 NN denotes exon
T5791 7939-7941 CD denotes 10
T5792 7942-7944 IN denotes to
T5793 7945-7954 NNS denotes sequences
T5794 7955-7965 RB denotes downstream
T5795 7966-7968 IN denotes of
T5796 7969-7972 DT denotes the
T5797 7973-7977 NN denotes gene
T5798 7977-7978 -RRB- denotes )
T5799 7979-7983 IN denotes into
T5800 7984-7987 NN denotes pBS
T5801 7987-7989 , denotes ,
T5802 7989-7993 RB denotes then
T5803 7994-8001 VBD denotes mutated
T5804 8002-8005 DT denotes the
T5806 8006-8013 NN denotes HindIII
T5805 8014-8018 NN denotes site
T5807 8019-8023 IN denotes into
T5808 8024-8025 DT denotes a
T5810 8026-8030 NN denotes FseI
T5809 8031-8035 NN denotes site
T5811 8035-8036 . denotes .
T5812 8036-8427 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 8037-8039 PRP denotes We
T5815 8040-8044 RB denotes next
T5814 8045-8052 VBD denotes mutated
T5816 8053-8056 DT denotes the
T5818 8057-8058 NN denotes C
T5820 8058-8059 HYPH denotes -
T5819 8059-8067 JJ denotes terminal
T5817 8068-8072 NN denotes part
T5821 8073-8075 IN denotes of
T5822 8076-8079 DT denotes the
T5824 8080-8083 NN denotes p53
T5823 8084-8088 NN denotes gene
T5825 8089-8091 IN denotes in
T5826 8092-8095 CD denotes two
T5827 8096-8102 NNS denotes rounds
T5828 8103-8105 IN denotes of
T5829 8106-8109 NN denotes PCR
T5830 8110-8121 NN denotes mutagenesis
T5831 8121-8123 , denotes ,
T5832 8123-8128 RB denotes first
T5833 8129-8133 IN denotes with
T5834 8134-8141 NNS denotes primers
T5836 8142-8143 CD denotes 5
T5837 8143-8144 SYM denotes ′
T5838 8144-8145 HYPH denotes -
T5835 8145-8181 NN denotes GGGCCTGACTCAGACGGATCCCCTCTGCATCCCGTC
T5839 8181-8182 HYPH denotes -
T5840 8182-8183 CD denotes 3
T5841 8183-8184 SYM denotes ′
T5842 8185-8188 CC denotes and
T5843 8189-8190 CD denotes 5
T5845 8190-8191 SYM denotes ′
T5846 8191-8192 HYPH denotes -
T5844 8192-8228 NN denotes GACGGGATGCAGAGGGGATCCGTCTGAGTCAGGCCC
T5847 8228-8229 HYPH denotes -
T5848 8229-8230 CD denotes 3
T5849 8230-8231 SYM denotes ′
T5850 8231-8233 , denotes ,
T5851 8233-8238 WDT denotes which
T5852 8239-8246 VBD denotes removed
T5853 8247-8250 DT denotes the
T5855 8251-8255 NN denotes stop
T5854 8256-8261 NN denotes codon
T5856 8262-8265 CC denotes and
T5857 8266-8276 VBD denotes introduced
T5858 8277-8278 DT denotes a
T5860 8279-8284 NN denotes BamHI
T5859 8285-8289 NN denotes site
T5861 8289-8291 , denotes ,
T5862 8291-8295 RB denotes then
T5863 8296-8300 IN denotes with
T5864 8301-8308 NNS denotes primers
T5866 8309-8310 CD denotes 5
T5867 8310-8311 SYM denotes ′
T5868 8311-8312 HYPH denotes -
T5865 8312-8346 NN denotes GACGGATCCCCTCTGAATTCCGTCCCCATCACCA
T5869 8346-8347 HYPH denotes -
T5870 8347-8348 CD denotes 3
T5871 8348-8349 SYM denotes ′
T5872 8350-8353 CC denotes and
T5873 8354-8355 CD denotes 5
T5875 8355-8356 SYM denotes ′
T5876 8356-8357 HYPH denotes -
T5874 8357-8391 NN denotes TGGTGATGGGGACGGAATACAGAGGGGATCCGTC
T5877 8391-8392 HYPH denotes -
T5878 8392-8393 CD denotes 3
T5879 8393-8394 SYM denotes ′
T5880 8394-8396 , denotes ,
T5881 8396-8401 WDT denotes which
T5882 8402-8412 VBD denotes introduced
T5883 8413-8415 DT denotes an
T5885 8416-8421 NN denotes EcoRI
T5884 8422-8426 NN denotes site
T5886 8426-8427 . denotes .
T5887 8427-8614 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 8428-8430 PRP denotes We
T5889 8431-8439 VBD denotes verified
T5890 8440-8443 DT denotes the
T5891 8444-8452 NN denotes sequence
T5892 8453-8457 IN denotes from
T5893 8458-8461 DT denotes the
T5895 8462-8469 VBN denotes mutated
T5894 8470-8477 NN denotes plasmid
T5896 8477-8479 , denotes ,
T5897 8479-8483 RB denotes then
T5898 8484-8492 VBD denotes digested
T5899 8493-8495 PRP denotes it
T5900 8496-8500 IN denotes with
T5901 8501-8506 NN denotes BamHI
T5902 8507-8510 CC denotes and
T5903 8511-8516 NN denotes EcoRI
T5904 8516-8518 , denotes ,
T5905 8518-8520 TO denotes to
T5906 8521-8527 VB denotes insert
T5907 8528-8530 IN denotes in
T5908 8531-8536 NN denotes frame
T5909 8537-8540 NN denotes GFP
T5910 8541-8550 NNS denotes sequences
T5911 8551-8555 IN denotes from
T5912 8556-8557 DT denotes a
T5914 8558-8561 NN denotes Bam
T5916 8562-8564 NN denotes HI
T5917 8564-8565 HYPH denotes -
T5915 8565-8570 NN denotes EcoRI
T5913 8571-8579 NN denotes fragment
T5918 8580-8582 IN denotes of
T5919 8583-8590 NN denotes plasmid
T5921 8591-8594 NN denotes phr
T5922 8594-8595 HYPH denotes -
T5920 8595-8598 NN denotes GFP
T5923 8598-8599 HYPH denotes -
T5924 8599-8600 CD denotes 1
T5925 8601-8602 -LRB- denotes (
T5926 8602-8612 NNP denotes Stratagene
T5927 8612-8613 -RRB- denotes )
T5928 8613-8614 . denotes .
T5929 8614-8857 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 8615-8617 PRP denotes We
T5931 8618-8626 VBD denotes verified
T5932 8627-8630 DT denotes the
T5933 8631-8639 NN denotes sequence
T5934 8640-8642 IN denotes of
T5935 8643-8647 DT denotes this
T5937 8648-8651 NN denotes p53
T5939 8651-8652 HYPH denotes -
T5938 8652-8655 NN denotes GFP
T5940 8656-8662 NN denotes fusion
T5936 8663-8671 NN denotes fragment
T5941 8671-8673 , denotes ,
T5942 8673-8677 RB denotes then
T5943 8678-8685 VBD denotes swapped
T5944 8686-8688 PRP denotes it
T5945 8689-8691 IN denotes in
T5946 8692-8695 DT denotes the
T5948 8696-8698 NN denotes L3
T5950 8698-8699 HYPH denotes -
T5951 8699-8715 NN denotes p53PmlEagPuroΔTK
T5952 8715-8716 HYPH denotes -
T5949 8716-8718 NN denotes 1L
T5947 8719-8726 NN denotes plasmid
T5953 8727-8728 -LRB- denotes (
T5954 8728-8731 VB denotes see
T5955 8732-8737 RB denotes above
T5956 8737-8738 -RRB- denotes )
T5957 8739-8741 IN denotes by
T5958 8742-8749 NN denotes HindIII
T5960 8750-8753 CC denotes and
T5961 8754-8758 NN denotes FseI
T5959 8759-8768 NN denotes digestion
T5962 8768-8770 , denotes ,
T5963 8770-8779 VBG denotes resulting
T5964 8780-8782 IN denotes in
T5965 8783-8786 DT denotes the
T5967 8787-8793 NN denotes p53GFP
T5968 8794-8802 NN denotes exchange
T5966 8803-8812 NN denotes construct
T5969 8812-8814 , denotes ,
T5970 8814-8817 DT denotes the
T5971 8818-8826 NN denotes sequence
T5972 8827-8832 WDT denotes which
T5974 8833-8836 VBD denotes was
T5973 8837-8845 VBN denotes verified
T5975 8846-8852 IN denotes before
T5976 8853-8856 NN denotes use
T5977 8856-8857 . denotes .
T5978 8857-9033 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 8858-8861 DT denotes The
T5981 8862-8870 NN denotes p53ΔPGFP
T5982 8871-8879 NN denotes exchange
T5980 8880-8889 NN denotes construct
T5984 8890-8893 VBD denotes was
T5983 8894-8904 VBN denotes engineered
T5985 8905-8907 IN denotes by
T5986 8908-8917 VBG denotes combining
T5987 8918-8927 NNS denotes sequences
T5988 8928-8932 IN denotes from
T5989 8933-8936 DT denotes the
T5991 8937-8943 NN denotes p53GFP
T5992 8944-8952 NN denotes exchange
T5990 8953-8960 NN denotes plasmid
T5993 8961-8964 CC denotes and
T5994 8965-8974 NNS denotes sequences
T5995 8975-8979 IN denotes from
T5996 8980-8983 DT denotes the
T5998 8984-8989 NN denotes p53ΔP
T5999 8990-8999 NN denotes targeting
T5997 9000-9009 NN denotes construct
T6000 9010-9019 VBN denotes described
T6001 9020-9028 RB denotes recently
T6002 9029-9030 -LRB- denotes (
T6003 9030-9031 CD denotes 5
T6004 9031-9032 -RRB- denotes )
T6005 9032-9033 . denotes .
T6006 9033-9076 sentence denotes Its sequence was also verified before use.
T6007 9034-9037 PRP$ denotes Its
T6008 9038-9046 NN denotes sequence
T6010 9047-9050 VBD denotes was
T6011 9051-9055 RB denotes also
T6009 9056-9064 VBN denotes verified
T6012 9065-9071 IN denotes before
T6013 9072-9075 NN denotes use
T6014 9075-9076 . denotes .
T6352 9078-9087 NNS denotes Sequences
T6353 9088-9091 CC denotes and
T6354 9092-9095 NN denotes use
T6355 9096-9098 IN denotes of
T6356 9099-9102 NN denotes PCR
T6357 9103-9110 NNS denotes primers
T6358 9110-9910 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 9111-9112 LS denotes a
T6361 9112-9114 : denotes :
T6362 9114-9115 CD denotes 5
T6363 9115-9116 SYM denotes ′
T6364 9116-9117 HYPH denotes -
T6360 9117-9141 NN denotes CCCCGGCCCTCACCCTCATCTTCG
T6366 9141-9142 HYPH denotes -
T6367 9142-9143 CD denotes 3
T6368 9143-9144 SYM denotes ′
T6369 9144-9146 , denotes ,
T6370 9146-9150 IN denotes from
T6371 9151-9154 DT denotes the
T6373 9155-9160 NN denotes PuΔTK
T6372 9161-9165 NN denotes gene
T6374 9165-9167 , denotes ,
T6375 9167-9173 NNS denotes assays
T6376 9174-9183 VBG denotes targeting
T6377 9184-9186 IN denotes of
T6378 9187-9191 NN denotes Flox
T6379 9192-9199 NN denotes plasmid
T6380 9199-9200 : denotes ;
T6381 9201-9202 LS denotes b
T6383 9202-9204 : denotes :
T6384 9204-9205 CD denotes 5
T6385 9205-9206 SYM denotes ′
T6386 9206-9207 HYPH denotes -
T6382 9207-9231 NN denotes AACAAAACAAAACAGCAGCAACAA
T6387 9231-9232 HYPH denotes -
T6388 9232-9233 CD denotes 3
T6389 9233-9234 SYM denotes ′
T6390 9234-9236 , denotes ,
T6391 9236-9240 IN denotes from
T6392 9241-9250 NNS denotes sequences
T6393 9251-9261 RB denotes downstream
T6395 9262-9264 IN denotes of
T6396 9265-9268 DT denotes the
T6398 9269-9272 NN denotes p53
T6397 9273-9277 NN denotes gene
T6399 9278-9281 CC denotes and
T6394 9282-9289 IN denotes outside
T6400 9290-9294 NN denotes Flox
T6401 9295-9304 NNS denotes sequences
T6402 9304-9306 , denotes ,
T6403 9306-9312 NNS denotes assays
T6404 9313-9322 VBG denotes targeting
T6405 9323-9325 IN denotes of
T6406 9326-9330 NN denotes Flox
T6407 9331-9334 CC denotes and
T6408 9335-9339 NN denotes RMCE
T6409 9340-9344 IN denotes with
T6410 9345-9351 NN denotes p53GFP
T6412 9352-9354 CC denotes or
T6413 9355-9363 NN denotes p53ΔPGFP
T6411 9364-9372 NNS denotes plasmids
T6414 9372-9373 : denotes ;
T6415 9374-9375 LS denotes c
T6417 9375-9377 : denotes :
T6418 9377-9378 CD denotes 5
T6419 9378-9379 SYM denotes ′
T6420 9379-9380 HYPH denotes -
T6416 9380-9405 NN denotes TGAAGAGCAAGGGCGTGGTGAAGGA
T6421 9405-9406 HYPH denotes -
T6422 9406-9407 CD denotes 3
T6423 9407-9408 SYM denotes ′
T6424 9408-9410 , denotes ,
T6425 9410-9414 IN denotes from
T6426 9415-9418 NN denotes GFP
T6427 9419-9428 NNS denotes sequences
T6428 9428-9430 , denotes ,
T6429 9430-9436 NNS denotes assays
T6430 9437-9441 NN denotes RMCE
T6431 9442-9446 IN denotes with
T6432 9447-9453 NN denotes p53GFP
T6433 9454-9464 NN denotes orp53ΔPGFP
T6434 9465-9473 NNS denotes plasmids
T6435 9473-9474 : denotes ;
T6436 9475-9476 LS denotes d
T6438 9476-9478 : denotes :
T6439 9478-9479 CD denotes 5
T6440 9479-9480 SYM denotes ′
T6441 9480-9481 HYPH denotes -
T6437 9481-9509 NN denotes CAAAAAATGGAAGGAAATCAGGAACTAA
T6442 9509-9510 HYPH denotes -
T6443 9510-9511 CD denotes 3
T6444 9511-9512 SYM denotes ′
T6445 9512-9514 , denotes ,
T6446 9514-9518 IN denotes from
T6447 9519-9522 NN denotes p53
T6448 9523-9529 NN denotes intron
T6449 9530-9531 CD denotes 3
T6450 9531-9533 , denotes ,
T6451 9533-9536 CC denotes and
T6452 9537-9538 LS denotes e
T6454 9538-9540 : denotes :
T6455 9540-9541 CD denotes 5
T6456 9541-9542 SYM denotes ′
T6457 9542-9543 HYPH denotes -
T6453 9543-9568 NN denotes TCTAGACAGAGAAAAAAGAGGCATT
T6458 9568-9569 HYPH denotes -
T6459 9569-9570 CD denotes 3
T6460 9570-9571 SYM denotes ′
T6461 9571-9573 , denotes ,
T6462 9573-9577 IN denotes from
T6463 9578-9581 NN denotes p53
T6464 9582-9588 NN denotes intron
T6465 9589-9590 CD denotes 4
T6466 9590-9592 , denotes ,
T6467 9592-9597 NN denotes assay
T6468 9598-9602 NN denotes RMCE
T6469 9603-9607 IN denotes with
T6470 9608-9616 NN denotes p53ΔPGFP
T6471 9617-9624 NN denotes plasmid
T6472 9624-9625 : denotes ;
T6473 9626-9627 LS denotes f
T6475 9627-9629 : denotes :
T6476 9629-9630 CD denotes 5
T6477 9630-9631 SYM denotes ′
T6478 9631-9632 HYPH denotes -
T6474 9632-9656 NN denotes ATGGGAGGCTGCCAGTCCTAACCC
T6479 9657-9660 CC denotes and
T6480 9661-9662 LS denotes g
T6482 9662-9664 : denotes :
T6483 9664-9665 CD denotes 5
T6484 9665-9666 SYM denotes ′
T6485 9666-9667 HYPH denotes -
T6481 9667-9693 NN denotes GTGTTTCATTAGTTCCCCACCTTGAC
T6486 9693-9694 HYPH denotes -
T6487 9694-9695 CD denotes 3
T6488 9695-9696 SYM denotes ′
T6365 9697-9704 VBP denotes amplify
T6490 9705-9708 DT denotes the
T6492 9709-9711 NN denotes WT
T6493 9712-9715 NN denotes p53
T6491 9716-9722 NN denotes allele
T6494 9723-9732 IN denotes according
T6495 9733-9735 IN denotes to
T6496 9736-9743 NNP denotes Taconic
T6498 9743-9745 POS denotes 's
T6497 9746-9756 NNS denotes procedures
T6499 9756-9758 , denotes ,
T6500 9758-9759 LS denotes h
T6502 9759-9761 : denotes :
T6503 9761-9762 CD denotes 5
T6504 9762-9763 SYM denotes ′
T6505 9763-9764 HYPH denotes -
T6501 9764-9789 NN denotes TTTACGGAGCCCTGGCGCTCGATGT
T6506 9789-9790 HYPH denotes -
T6507 9790-9791 CD denotes 3
T6508 9791-9792 SYM denotes ′
T6509 9793-9796 CC denotes and
T6510 9797-9798 LS denotes i
T6512 9798-9800 : denotes :
T6513 9800-9801 CD denotes 5
T6514 9801-9802 SYM denotes ′
T6515 9802-9803 HYPH denotes -
T6511 9803-9828 NN denotes GTGGGAGGGACAAAAGTTCGAGGCC
T6516 9828-9829 HYPH denotes -
T6517 9829-9830 CD denotes 3
T6518 9830-9831 SYM denotes ′
T6489 9832-9839 VBP denotes amplify
T6519 9840-9843 DT denotes the
T6521 9844-9847 NN denotes Neo
T6520 9848-9854 NN denotes marker
T6522 9855-9857 IN denotes in
T6523 9858-9861 DT denotes the
T6525 9862-9865 NN denotes p53
T6526 9866-9868 NN denotes KO
T6524 9869-9875 NN denotes allele
T6527 9876-9885 IN denotes according
T6528 9886-9888 IN denotes to
T6529 9889-9896 NNP denotes Taconic
T6531 9896-9898 POS denotes 's
T6530 9899-9909 NNS denotes procedures
T6532 9909-9910 . denotes .
T6727 9912-9916 NN denotes Cell
T6728 9917-9924 NN denotes culture
T6729 9925-9935 NNS denotes conditions
T6730 9935-10063 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 9936-9943 JJ denotes Primary
T6732 9944-9948 NNS denotes MEFs
T6734 9948-9950 , denotes ,
T6735 9950-9958 VBN denotes isolated
T6736 9959-9963 IN denotes from
T6737 9964-9968 CD denotes 13.5
T6738 9969-9972 NN denotes day
T6739 9973-9980 NNS denotes embryos
T6740 9980-9982 , denotes ,
T6741 9982-9986 VBD denotes were
T6733 9987-9995 VBN denotes cultured
T6742 9996-9998 IN denotes in
T6743 9999-10003 NN denotes DMEM
T6744 10004-10008 IN denotes with
T6745 10009-10011 CD denotes 15
T6746 10011-10012 NN denotes %
T6747 10013-10016 NN denotes FBS
T6748 10016-10018 , denotes ,
T6749 10018-10021 CD denotes 100
T6750 10022-10024 NN denotes mM
T6751 10025-10028 NN denotes BME
T6752 10028-10030 , denotes ,
T6753 10030-10031 CD denotes 2
T6754 10032-10034 NN denotes mM
T6756 10035-10036 NN denotes l
T6757 10036-10037 HYPH denotes -
T6755 10037-10046 NN denotes glutamine
T6758 10047-10050 CC denotes and
T6759 10051-10062 NNS denotes antibiotics
T6760 10062-10063 . denotes .
T6761 10063-10239 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 10064-10067 CD denotes 129
T6764 10067-10068 HYPH denotes /
T6763 10068-10073 NN denotes SvJae
T6766 10074-10076 NN denotes ES
T6765 10077-10082 NNS denotes cells
T6768 10083-10087 VBD denotes were
T6767 10088-10093 VBN denotes grown
T6769 10094-10096 IN denotes in
T6770 10097-10100 DT denotes the
T6772 10101-10105 JJ denotes same
T6771 10106-10112 NN denotes medium
T6773 10113-10125 VBN denotes supplemented
T6774 10126-10130 IN denotes with
T6775 10131-10135 CD denotes 1000
T6776 10136-10137 NN denotes U
T6778 10137-10138 SYM denotes /
T6779 10138-10140 NN denotes ml
T6777 10141-10146 NN denotes ESGRO
T6780 10147-10148 -LRB- denotes (
T6781 10148-10156 NNP denotes Chemicon
T6782 10156-10157 -RRB- denotes )
T6783 10157-10159 , denotes ,
T6784 10159-10161 IN denotes on
T6785 10162-10163 DT denotes a
T6786 10164-10169 NN denotes layer
T6787 10170-10172 IN denotes of
T6788 10173-10182 NN denotes mitomycin
T6789 10183-10184 NN denotes C
T6791 10184-10185 HYPH denotes -
T6790 10185-10192 JJ denotes treated
T6793 10193-10200 NN denotes SNLPuro
T6794 10200-10201 HYPH denotes -
T6795 10201-10202 CD denotes 7
T6796 10202-10203 HYPH denotes /
T6797 10203-10204 CD denotes 4
T6792 10205-10212 NNS denotes feeders
T6798 10213-10214 -LRB- denotes (
T6800 10214-10218 JJ denotes kind
T6799 10219-10223 NN denotes gift
T6801 10224-10226 IN denotes of
T6802 10227-10229 NNP denotes A.
T6803 10230-10237 NNP denotes Bradley
T6804 10237-10238 -RRB- denotes )
T6805 10238-10239 . denotes .
T6806 10239-10322 sentence denotes Selections were performed with 2 μg/ml puromycin, 0.2 μM FIAU or 2 μM ganciclovir.
T6807 10240-10250 NNS denotes Selections
T6809 10251-10255 VBD denotes were
T6808 10256-10265 VBN denotes performed
T6810 10266-10270 IN denotes with
T6811 10271-10272 CD denotes 2
T6812 10273-10275 NN denotes μg
T6814 10275-10276 SYM denotes /
T6815 10276-10278 NN denotes ml
T6813 10279-10288 NN denotes puromycin
T6816 10288-10290 , denotes ,
T6817 10290-10293 CD denotes 0.2
T6818 10294-10296 NN denotes μM
T6819 10297-10301 NN denotes FIAU
T6820 10302-10304 CC denotes or
T6821 10305-10306 CD denotes 2
T6822 10307-10309 NN denotes μM
T6823 10310-10321 NN denotes ganciclovir
T6824 10321-10322 . denotes .
T6929 10324-10333 NN denotes Targeting
T6931 10333-10334 HYPH denotes /
T6930 10334-10344 NN denotes genotyping
T6932 10345-10347 IN denotes of
T6933 10348-10351 DT denotes the
T6935 10352-10356 JJ denotes RMCE
T6937 10356-10357 HYPH denotes -
T6936 10357-10362 JJ denotes ready
T6934 10363-10368 NN denotes locus
T6938 10368-10522 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 10369-10371 CD denotes 29
T6941 10371-10372 HYPH denotes /
T6940 10372-10377 NN denotes SvJae
T6943 10378-10380 NN denotes ES
T6942 10381-10386 NNS denotes cells
T6945 10387-10391 VBD denotes were
T6944 10392-10406 VBN denotes electroporated
T6946 10407-10411 IN denotes with
T6947 10412-10415 DT denotes the
T6949 10416-10420 NN denotes Flox
T6948 10421-10430 NN denotes construct
T6950 10431-10441 VBN denotes linearized
T6951 10442-10446 IN denotes with
T6952 10447-10451 NN denotes PmeI
T6953 10451-10453 , denotes ,
T6954 10453-10456 CC denotes and
T6955 10457-10466 NN denotes puromycin
T6956 10467-10476 JJ denotes resistant
T6957 10477-10483 NNS denotes clones
T6959 10484-10488 VBD denotes were
T6958 10489-10497 VBN denotes analyzed
T6960 10498-10500 IN denotes as
T6961 10501-10510 VBN denotes described
T6962 10511-10512 -LRB- denotes (
T6963 10512-10518 NN denotes Figure
T6964 10519-10520 CD denotes 2
T6965 10520-10521 -RRB- denotes )
T6966 10521-10522 . denotes .
T6967 10522-10602 sentence denotes Two clones were injected into blastocysts and transmitted through the germline.
T6968 10523-10526 CD denotes Two
T6969 10527-10533 NNS denotes clones
T6971 10534-10538 VBD denotes were
T6970 10539-10547 VBN denotes injected
T6972 10548-10552 IN denotes into
T6973 10553-10564 NNS denotes blastocysts
T6974 10565-10568 CC denotes and
T6975 10569-10580 VBN denotes transmitted
T6976 10581-10588 IN denotes through
T6977 10589-10592 DT denotes the
T6978 10593-10601 NN denotes germline
T6979 10601-10602 . denotes .
T7192 10604-10614 VBG denotes Performing
T7193 10615-10619 NN denotes RMCE
T7194 10620-10622 IN denotes in
T7195 10623-10625 NN denotes ES
T7196 10626-10631 NNS denotes cells
T7197 10631-10846 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 10632-10633 DT denotes A
T7199 10634-10639 NN denotes total
T7201 10640-10642 IN denotes of
T7202 10643-10644 CD denotes 8
T7204 10645-10646 HYPH denotes ×
T7203 10647-10650 CD denotes 105
T7206 10651-10658 NN denotes p53RMCE
T7207 10658-10659 HYPH denotes /
T7208 10659-10660 SYM denotes +
T7209 10661-10663 NN denotes ES
T7205 10664-10669 NNS denotes cells
T7210 10670-10674 VBD denotes were
T7200 10675-10680 VBN denotes grown
T7211 10681-10688 IN denotes without
T7212 10689-10698 NN denotes puromycin
T7213 10699-10702 IN denotes for
T7214 10703-10705 CD denotes 12
T7215 10706-10707 NN denotes h
T7216 10707-10709 , denotes ,
T7217 10709-10723 VBN denotes electroporated
T7218 10724-10728 IN denotes with
T7219 10729-10731 CD denotes 15
T7221 10732-10734 NN denotes μg
T7222 10735-10738 NN denotes CMV
T7224 10738-10739 HYPH denotes -
T7223 10739-10742 NN denotes Cre
T7220 10743-10750 NN denotes plasmid
T7225 10751-10752 -LRB- denotes (
T7226 10752-10758 NN denotes pOG231
T7227 10758-10759 -RRB- denotes )
T7228 10760-10763 CC denotes and
T7229 10764-10767 CD denotes 200
T7230 10768-10770 NN denotes μg
T7231 10771-10773 IN denotes of
T7232 10774-10777 DT denotes the
T7234 10778-10786 NN denotes exchange
T7233 10787-10796 NN denotes construct
T7235 10796-10798 , denotes ,
T7236 10798-10801 CC denotes and
T7237 10802-10808 VBN denotes plated
T7238 10809-10811 IN denotes in
T7239 10812-10815 NN denotes T25
T7240 10816-10822 NNS denotes flasks
T7241 10823-10825 IN denotes at
T7242 10826-10829 CD denotes 105
T7243 10830-10835 NNS denotes cells
T7244 10836-10839 IN denotes per
T7245 10840-10845 NN denotes flask
T7246 10845-10846 . denotes .
T7247 10846-10907 sentence denotes FIAU was added to the medium 3–4 days after electroporation.
T7248 10847-10851 NN denotes FIAU
T7250 10852-10855 VBD denotes was
T7249 10856-10861 VBN denotes added
T7251 10862-10864 IN denotes to
T7252 10865-10868 DT denotes the
T7253 10869-10875 NN denotes medium
T7254 10876-10877 CD denotes 3
T7256 10877-10878 HYPH denotes –
T7255 10878-10879 CD denotes 4
T7257 10880-10884 NNS denotes days
T7258 10885-10890 IN denotes after
T7259 10891-10906 NN denotes electroporation
T7260 10906-10907 . denotes .
T7261 10907-11086 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 10908-10918 JJ denotes Individual
T7263 10919-10925 NNS denotes clones
T7265 10925-10927 , denotes ,
T7266 10927-10933 VBN denotes picked
T7267 10934-10936 CD denotes 10
T7268 10937-10941 NNS denotes days
T7269 10942-10947 IN denotes after
T7270 10948-10963 NN denotes electroporation
T7271 10963-10965 , denotes ,
T7272 10965-10969 VBD denotes were
T7264 10970-10975 VBN denotes grown
T7273 10976-10978 IN denotes in
T7274 10979-10981 CD denotes 96
T7276 10981-10982 HYPH denotes -
T7275 10982-10986 NN denotes well
T7277 10987-10993 NNS denotes plates
T7278 10994-10997 CC denotes and
T7279 10998-11006 VBN denotes expanded
T7280 11007-11009 TO denotes to
T7281 11010-11018 VB denotes generate
T7282 11019-11028 JJ denotes duplicate
T7283 11029-11035 NNS denotes plates
T7284 11036-11039 IN denotes for
T7285 11040-11048 VBG denotes freezing
T7286 11049-11052 CC denotes and
T7287 11053-11056 NN denotes DNA
T7288 11057-11065 NN denotes analysis
T7289 11066-11068 IN denotes by
T7290 11069-11072 NN denotes PCR
T7291 11073-11076 CC denotes and
T7292 11077-11085 NNP denotes Southern
T7293 11085-11086 . denotes .
T7497 11088-11098 VBG denotes Performing
T7498 11099-11103 NN denotes RMCE
T7499 11104-11106 IN denotes in
T7500 11107-11111 NNS denotes MEFs
T7501 11111-11352 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 11112-11113 DT denotes A
T7503 11114-11119 NN denotes total
T7505 11120-11122 IN denotes of
T7506 11123-11126 CD denotes 106
T7508 11127-11134 NN denotes p53RMCE
T7510 11134-11135 HYPH denotes /
T7511 11135-11136 SYM denotes −
T7509 11137-11141 NNS denotes MEFs
T7507 11142-11147 NNS denotes cells
T7512 11148-11152 VBD denotes were
T7504 11153-11158 VBN denotes grown
T7513 11159-11166 IN denotes without
T7514 11167-11176 NN denotes puromycin
T7515 11177-11180 IN denotes for
T7516 11181-11183 CD denotes 12
T7517 11184-11185 NN denotes h
T7518 11185-11187 , denotes ,
T7519 11187-11201 VBN denotes electroporated
T7520 11202-11206 IN denotes with
T7521 11207-11208 CD denotes 3
T7522 11209-11211 NN denotes μg
T7523 11212-11218 NN denotes pOG231
T7524 11219-11222 CC denotes and
T7525 11223-11225 CD denotes 30
T7526 11226-11228 NN denotes μg
T7528 11229-11237 NN denotes exchange
T7527 11238-11247 NN denotes construct
T7529 11247-11249 , denotes ,
T7530 11249-11252 CC denotes and
T7531 11253-11259 VBN denotes plated
T7532 11260-11262 IN denotes in
T7533 11263-11264 DT denotes a
T7535 11265-11271 JJ denotes single
T7536 11272-11274 CD denotes 10
T7537 11275-11277 NN denotes cm
T7538 11277-11278 HYPH denotes -
T7534 11278-11282 NN denotes dish
T7539 11282-11284 , denotes ,
T7540 11284-11289 VBN denotes grown
T7541 11290-11293 IN denotes for
T7542 11294-11295 CD denotes 3
T7543 11296-11300 NNS denotes days
T7544 11301-11305 RB denotes then
T7545 11306-11311 VBN denotes split
T7546 11312-11314 IN denotes in
T7547 11315-11322 JJ denotes several
T7548 11323-11329 NNS denotes dishes
T7549 11330-11332 IN denotes at
T7550 11333-11336 CD denotes 105
T7551 11337-11342 NNS denotes cells
T7552 11343-11346 IN denotes per
T7553 11347-11351 NN denotes dish
T7554 11351-11352 . denotes .
T7555 11352-11440 sentence denotes FIAU or ganciclovir was added to the medium 4 days after electroporation, for 3–4 days.
T7556 11353-11357 NN denotes FIAU
T7558 11358-11360 CC denotes or
T7559 11361-11372 NN denotes ganciclovir
T7560 11373-11376 VBD denotes was
T7557 11377-11382 VBN denotes added
T7561 11383-11385 IN denotes to
T7562 11386-11389 DT denotes the
T7563 11390-11396 NN denotes medium
T7564 11397-11398 CD denotes 4
T7565 11399-11403 NNS denotes days
T7566 11404-11409 IN denotes after
T7567 11410-11425 NN denotes electroporation
T7568 11425-11427 , denotes ,
T7569 11427-11430 IN denotes for
T7570 11431-11432 CD denotes 3
T7572 11432-11433 SYM denotes –
T7571 11433-11434 CD denotes 4
T7573 11435-11439 NNS denotes days
T7574 11439-11440 . denotes .
T7575 11440-11559 sentence denotes Clones, picked 10 days after electroporation, were grown in 24-well plates and expanded for freezing and DNA analysis.
T7576 11441-11447 NNS denotes Clones
T7578 11447-11449 , denotes ,
T7579 11449-11455 VBN denotes picked
T7580 11456-11458 CD denotes 10
T7581 11459-11463 NNS denotes days
T7582 11464-11469 IN denotes after
T7583 11470-11485 NN denotes electroporation
T7584 11485-11487 , denotes ,
T7585 11487-11491 VBD denotes were
T7577 11492-11497 VBN denotes grown
T7586 11498-11500 IN denotes in
T7587 11501-11503 CD denotes 24
T7589 11503-11504 HYPH denotes -
T7588 11504-11508 NN denotes well
T7590 11509-11515 NNS denotes plates
T7591 11516-11519 CC denotes and
T7592 11520-11528 VBN denotes expanded
T7593 11529-11532 IN denotes for
T7594 11533-11541 NN denotes freezing
T7595 11542-11545 CC denotes and
T7596 11546-11549 NN denotes DNA
T7597 11550-11558 NN denotes analysis
T7598 11558-11559 . denotes .
T8070 11561-11568 NNP denotes Western
T8072 11568-11569 HYPH denotes -
T8071 11569-11574 NNS denotes blots
T8073 11574-11871 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 11575-11580 NNS denotes Cells
T8076 11580-11582 , denotes ,
T8077 11582-11591 JJ denotes untreated
T8078 11592-11594 CC denotes or
T8079 11595-11602 VBN denotes treated
T8080 11603-11606 IN denotes for
T8081 11607-11609 CD denotes 24
T8082 11610-11611 NN denotes h
T8083 11612-11616 IN denotes with
T8084 11617-11620 CD denotes 0.5
T8085 11621-11623 NN denotes μg
T8087 11623-11624 SYM denotes /
T8088 11624-11626 NN denotes ml
T8086 11627-11637 NN denotes adriamycin
T8089 11637-11639 , denotes ,
T8090 11639-11643 VBD denotes were
T8075 11644-11649 VBN denotes lysed
T8091 11650-11652 IN denotes on
T8092 11653-11656 DT denotes the
T8093 11657-11661 NN denotes dish
T8094 11662-11664 IN denotes in
T8095 11665-11666 DT denotes a
T8096 11667-11673 NN denotes buffer
T8097 11674-11684 VBG denotes consisting
T8098 11685-11687 IN denotes of
T8099 11688-11690 CD denotes 50
T8100 11691-11693 NN denotes mM
T8101 11694-11698 NN denotes Tris
T8102 11699-11700 -LRB- denotes (
T8103 11700-11702 NN denotes pH
T8104 11703-11706 CD denotes 8.0
T8105 11706-11707 -RRB- denotes )
T8106 11707-11709 , denotes ,
T8107 11709-11710 CD denotes 5
T8108 11711-11713 NN denotes mM
T8109 11714-11718 NN denotes EDTA
T8110 11718-11720 , denotes ,
T8111 11720-11723 CD denotes 150
T8112 11724-11726 NN denotes mM
T8113 11727-11731 NN denotes NaCl
T8114 11731-11733 , denotes ,
T8115 11733-11736 CD denotes 0.5
T8116 11736-11737 NN denotes %
T8118 11738-11745 NN denotes Nonidet
T8119 11746-11747 NN denotes P
T8120 11747-11748 HYPH denotes -
T8117 11748-11750 NN denotes 40
T8121 11750-11752 , denotes ,
T8122 11752-11753 CD denotes 1
T8123 11754-11756 NN denotes mM
T8124 11757-11761 NN denotes PMSF
T8125 11761-11763 , denotes ,
T8126 11763-11764 CD denotes 1
T8127 11765-11767 NN denotes mM
T8129 11768-11774 NN denotes sodium
T8128 11775-11783 NN denotes vanadate
T8130 11783-11785 , denotes ,
T8131 11785-11787 CD denotes 10
T8132 11788-11790 NN denotes mM
T8133 11791-11794 NNP denotes NaF
T8134 11795-11798 CC denotes and
T8135 11799-11807 NN denotes Complete
T8136 11808-11812 NN denotes Mini
T8138 11813-11821 NN denotes Protease
T8137 11822-11832 NNS denotes Inhibitors
T8139 11833-11834 -LRB- denotes (
T8141 11834-11839 NNP denotes Roche
T8140 11840-11851 NNP denotes Diagnostics
T8142 11851-11852 -RRB- denotes )
T8143 11853-11855 IN denotes at
T8144 11856-11857 CD denotes 4
T8145 11857-11859 NNS denotes °C
T8146 11860-11863 IN denotes for
T8147 11864-11866 CD denotes 30
T8148 11867-11870 NN denotes min
T8149 11870-11871 . denotes .
T8150 11871-11933 sentence denotes Lysates were scraped, then spun at 6000× g at 4°C for 10 min.
T8151 11872-11879 NNS denotes Lysates
T8153 11880-11884 VBD denotes were
T8152 11885-11892 VBN denotes scraped
T8154 11892-11894 , denotes ,
T8155 11894-11898 RB denotes then
T8156 11899-11903 VBN denotes spun
T8157 11904-11906 IN denotes at
T8158 11907-11911 CD denotes 6000
T8160 11911-11912 SYM denotes ×
T8159 11913-11914 NN denotes g
T8161 11915-11917 IN denotes at
T8162 11918-11919 CD denotes 4
T8163 11919-11921 NNS denotes °C
T8164 11922-11925 IN denotes for
T8165 11926-11928 CD denotes 10
T8166 11929-11932 NN denotes min
T8167 11932-11933 . denotes .
T8168 11933-12025 sentence denotes Protein concentration in the supernatant was determined using the Bio-Rad DC protein assay.
T8169 11934-11941 NN denotes Protein
T8170 11942-11955 NN denotes concentration
T8172 11956-11958 IN denotes in
T8173 11959-11962 DT denotes the
T8174 11963-11974 NN denotes supernatant
T8175 11975-11978 VBD denotes was
T8171 11979-11989 VBN denotes determined
T8176 11990-11995 VBG denotes using
T8177 11996-11999 DT denotes the
T8179 12000-12003 NN denotes Bio
T8181 12003-12004 HYPH denotes -
T8180 12004-12007 NN denotes Rad
T8182 12008-12010 NN denotes DC
T8183 12011-12018 NN denotes protein
T8178 12019-12024 NN denotes assay
T8184 12024-12025 . denotes .
T8185 12025-12180 sentence denotes Lysates were separated on single percentage SDS/PAGE gels, then electrophoretically transferred to poly(vinylidene difluoride), using standard procedures.
T8186 12026-12033 NNS denotes Lysates
T8188 12034-12038 VBD denotes were
T8187 12039-12048 VBN denotes separated
T8189 12049-12051 IN denotes on
T8190 12052-12058 JJ denotes single
T8191 12059-12069 NN denotes percentage
T8193 12070-12073 NN denotes SDS
T8195 12073-12074 HYPH denotes /
T8194 12074-12078 NN denotes PAGE
T8192 12079-12083 NNS denotes gels
T8196 12083-12085 , denotes ,
T8197 12085-12089 RB denotes then
T8199 12090-12109 RB denotes electrophoretically
T8198 12110-12121 VBN denotes transferred
T8200 12122-12124 IN denotes to
T8201 12125-12129 NN denotes poly
T8202 12129-12130 -LRB- denotes (
T8204 12130-12140 NN denotes vinylidene
T8203 12141-12151 NN denotes difluoride
T8205 12151-12152 -RRB- denotes )
T8206 12152-12154 , denotes ,
T8207 12154-12159 VBG denotes using
T8208 12160-12168 JJ denotes standard
T8209 12169-12179 NNS denotes procedures
T8210 12179-12180 . denotes .
T8211 12180-12185 NNS denotes Blots
T8213 12180-12399 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 12186-12190 VBD denotes were
T8212 12191-12200 VBN denotes incubated
T8215 12201-12203 IN denotes in
T8216 12204-12205 CD denotes 5
T8217 12205-12206 NN denotes %
T8219 12207-12214 JJ denotes non-fat
T8220 12215-12220 VBN denotes dried
T8218 12221-12225 NN denotes milk
T8221 12226-12228 IN denotes in
T8222 12229-12233 NN denotes TBST
T8223 12234-12235 -LRB- denotes (
T8225 12235-12239 CD denotes 0.02
T8226 12240-12241 NN denotes M
T8227 12242-12246 NN denotes Tris
T8228 12246-12248 , denotes ,
T8229 12248-12250 NN denotes pH
T8230 12251-12254 CD denotes 7.6
T8231 12254-12255 HYPH denotes /
T8232 12255-12259 CD denotes 0.35
T8233 12260-12261 NN denotes M
T8234 12262-12266 NN denotes NaCl
T8235 12266-12267 HYPH denotes /
T8236 12267-12270 CD denotes 0.1
T8237 12270-12271 NN denotes %
T8224 12272-12277 NN denotes Tween
T8238 12277-12278 HYPH denotes -
T8239 12278-12280 CD denotes 20
T8240 12280-12281 -RRB- denotes )
T8241 12282-12285 IN denotes for
T8242 12286-12287 CD denotes 1
T8243 12288-12289 NN denotes h
T8244 12290-12292 IN denotes at
T8245 12293-12297 NN denotes room
T8246 12298-12309 NN denotes temperature
T8247 12310-12316 IN denotes before
T8248 12317-12324 VBG denotes probing
T8249 12325-12329 IN denotes with
T8250 12330-12337 JJ denotes primary
T8251 12338-12348 NNS denotes antibodies
T8252 12349-12356 IN denotes against
T8253 12357-12360 NN denotes p53
T8254 12361-12362 -LRB- denotes (
T8256 12362-12364 NN denotes CM
T8257 12364-12365 HYPH denotes -
T8258 12365-12366 CD denotes 5
T8259 12366-12368 , denotes ,
T8255 12368-12378 NNP denotes Novacastra
T8260 12378-12379 -RRB- denotes )
T8261 12380-12383 CC denotes and
T8262 12384-12385 HYPH denotes -
T8263 12385-12390 NN denotes actin
T8264 12391-12392 -LRB- denotes (
T8265 12392-12397 NNP denotes Sigma
T8266 12397-12398 -RRB- denotes )
T8267 12398-12399 . denotes .
T8268 12399-12505 sentence denotes Secondary antibodies used include peroxidase-conjugated goat anti-mouse IgG and anti-rabbit IgG (Pierce).
T8269 12400-12409 JJ denotes Secondary
T8270 12410-12420 NNS denotes antibodies
T8272 12421-12425 VBN denotes used
T8271 12426-12433 VBP denotes include
T8273 12434-12444 NN denotes peroxidase
T8275 12444-12445 HYPH denotes -
T8274 12445-12455 VBN denotes conjugated
T8277 12456-12460 NN denotes goat
T8278 12461-12471 NN denotes anti-mouse
T8276 12472-12475 NN denotes IgG
T8279 12476-12479 CC denotes and
T8280 12480-12491 NN denotes anti-rabbit
T8281 12492-12495 NN denotes IgG
T8282 12496-12497 -LRB- denotes (
T8283 12497-12503 NNP denotes Pierce
T8284 12503-12504 -RRB- denotes )
T8285 12504-12505 . denotes .
T8286 12505-12622 sentence denotes Probed blots were incubated with Pierce Supersignal West Pico chemiluminescent substrate and exposed to X-ray films.
T8287 12506-12512 VBN denotes Probed
T8288 12513-12518 NNS denotes blots
T8290 12519-12523 VBD denotes were
T8289 12524-12533 VBN denotes incubated
T8291 12534-12538 IN denotes with
T8292 12539-12545 NNP denotes Pierce
T8294 12546-12557 NNP denotes Supersignal
T8295 12558-12562 NNP denotes West
T8296 12563-12567 NNP denotes Pico
T8297 12568-12584 JJ denotes chemiluminescent
T8293 12585-12594 NN denotes substrate
T8298 12595-12598 CC denotes and
T8299 12599-12606 VBN denotes exposed
T8300 12607-12609 IN denotes to
T8301 12610-12615 NN denotes X-ray
T8302 12616-12621 NNS denotes films
T8303 12621-12622 . denotes .
T8467 12624-12628 NN denotes Flow
T8468 12629-12638 NN denotes cytometry
T8469 12638-12749 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 12639-12642 NN denotes Log
T8472 12643-12648 NN denotes phase
T8471 12649-12654 NNS denotes cells
T8474 12655-12659 VBD denotes were
T8473 12660-12670 VBN denotes irradiated
T8475 12671-12673 IN denotes at
T8476 12674-12676 NN denotes RT
T8477 12677-12681 IN denotes with
T8478 12682-12683 DT denotes a
T8480 12684-12686 CD denotes 60
T8482 12687-12689 NN denotes Co
T8481 12690-12691 NN denotes γ
T8483 12691-12692 SYM denotes -
T8479 12692-12702 NN denotes irradiator
T8484 12703-12705 IN denotes at
T8485 12706-12711 NNS denotes doses
T8486 12712-12714 IN denotes of
T8487 12715-12716 CD denotes 6
T8489 12717-12719 CC denotes or
T8490 12720-12722 CD denotes 12
T8488 12723-12725 NN denotes Gy
T8491 12726-12729 CC denotes and
T8492 12730-12739 VBN denotes incubated
T8493 12740-12743 IN denotes for
T8494 12744-12746 CD denotes 24
T8495 12747-12748 NN denotes h
T8496 12748-12749 . denotes .
T8497 12749-12942 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 12750-12755 NNS denotes Cells
T8500 12756-12760 VBD denotes were
T8501 12761-12765 RB denotes then
T8502 12766-12771 NN denotes pulse
T8503 12771-12772 HYPH denotes -
T8499 12772-12779 VBN denotes labeled
T8504 12780-12783 IN denotes for
T8505 12784-12785 CD denotes 1
T8506 12786-12787 NN denotes h
T8507 12788-12792 IN denotes with
T8508 12793-12797 NN denotes BrdU
T8509 12798-12799 -LRB- denotes (
T8511 12799-12801 CD denotes 10
T8510 12802-12804 NN denotes μM
T8512 12804-12805 -RRB- denotes )
T8513 12805-12807 , denotes ,
T8514 12807-12812 VBN denotes fixed
T8515 12813-12815 IN denotes in
T8516 12816-12818 CD denotes 70
T8517 12818-12819 NN denotes %
T8518 12820-12827 NN denotes ethanol
T8519 12827-12829 , denotes ,
T8520 12829-12835 JJ denotes double
T8522 12835-12836 HYPH denotes -
T8521 12836-12843 VBN denotes stained
T8523 12844-12848 IN denotes with
T8524 12849-12853 NN denotes FITC
T8525 12854-12863 NN denotes anti-BrdU
T8526 12864-12867 CC denotes and
T8527 12868-12877 NN denotes propidium
T8528 12878-12884 NN denotes iodide
T8529 12884-12886 , denotes ,
T8530 12886-12890 RB denotes then
T8531 12891-12897 VBN denotes sorted
T8532 12898-12900 IN denotes by
T8533 12901-12906 VBG denotes using
T8534 12907-12908 DT denotes a
T8536 12909-12915 NNP denotes Becton
T8537 12916-12925 NNP denotes Dickinson
T8538 12926-12933 NNP denotes FACScan
T8535 12934-12941 NN denotes machine
T8539 12941-12942 . denotes .
T8540 12942-12999 sentence denotes Data were analyzed using Becton Dickinson Cellquest Pro.
T8541 12943-12947 NNS denotes Data
T8543 12948-12952 VBD denotes were
T8542 12953-12961 VBN denotes analyzed
T8544 12962-12967 VBG denotes using
T8545 12968-12974 NNP denotes Becton
T8546 12975-12984 NNP denotes Dickinson
T8548 12985-12994 NNP denotes Cellquest
T8547 12995-12998 NNP denotes Pro
T8549 12998-12999 . denotes .
T11673 13024-13027 DT denotes The
T11674 13028-13037 NN denotes rationale
T11676 13038-13041 IN denotes for
T11677 13042-13046 NN denotes RMCE
T11679 13046-13047 HYPH denotes -
T11678 13047-13051 NN denotes ASAP
T11680 13052-13054 VBZ denotes is
T11675 13055-13063 VBN denotes detailed
T11681 13064-13066 IN denotes in
T11682 13067-13073 NN denotes Figure
T11683 13074-13075 CD denotes 1
T11684 13075-13076 . denotes .
T11685 13076-13229 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 13077-13080 DT denotes The
T11688 13081-13086 JJ denotes first
T11687 13087-13091 NN denotes step
T11689 13092-13100 VBZ denotes requires
T11690 13101-13111 VBG denotes generating
T11691 13112-13113 DT denotes a
T11693 13114-13120 VBN denotes floxed
T11692 13121-13127 NN denotes allele
T11694 13128-13130 IN denotes in
T11695 13131-13133 NN denotes ES
T11696 13134-13139 NNS denotes cells
T11697 13140-13144 WDT denotes that
T11699 13145-13149 MD denotes will
T11698 13150-13155 VB denotes serve
T11700 13156-13158 IN denotes as
T11701 13159-13162 DT denotes the
T11702 13163-13172 NN denotes substrate
T11703 13173-13176 IN denotes for
T11704 13177-13187 JJ denotes subsequent
T11705 13188-13197 NNS denotes exchanges
T11706 13198-13199 -LRB- denotes (
T11708 13199-13203 NN denotes RMCE
T11710 13203-13204 HYPH denotes -
T11709 13204-13209 JJ denotes ready
T11712 13210-13212 NN denotes ES
T11711 13213-13217 NN denotes cell
T11713 13217-13219 , denotes ,
T11707 13219-13225 NN denotes Figure
T11714 13226-13227 CD denotes 1
T11715 13227-13228 -RRB- denotes )
T11716 13228-13229 . denotes .
T11717 13229-13277 sentence denotes The targeting strategy is detailed in Figure 2.
T11718 13230-13233 DT denotes The
T11720 13234-13243 VBG denotes targeting
T11719 13244-13252 NN denotes strategy
T11722 13253-13255 VBZ denotes is
T11721 13256-13264 VBN denotes detailed
T11723 13265-13267 IN denotes in
T11724 13268-13274 NN denotes Figure
T11725 13275-13276 CD denotes 2
T11726 13276-13277 . denotes .
T11727 13277-13404 sentence denotes The frequency of targeting was 4% (12/300 puromycin-resistant clones, analyzed by Southern blot and long-range PCR, Figure 2).
T11728 13278-13281 DT denotes The
T11729 13282-13291 NN denotes frequency
T11731 13292-13294 IN denotes of
T11732 13295-13304 NN denotes targeting
T11730 13305-13308 VBD denotes was
T11733 13309-13310 CD denotes 4
T11734 13310-13311 NN denotes %
T11735 13312-13313 -LRB- denotes (
T11737 13313-13315 CD denotes 12
T11739 13315-13316 HYPH denotes /
T11738 13316-13319 CD denotes 300
T11741 13320-13329 NN denotes puromycin
T11743 13329-13330 HYPH denotes -
T11742 13330-13339 JJ denotes resistant
T11740 13340-13346 NNS denotes clones
T11744 13346-13348 , denotes ,
T11745 13348-13356 VBN denotes analyzed
T11746 13357-13359 IN denotes by
T11747 13360-13368 NNP denotes Southern
T11748 13369-13373 NN denotes blot
T11749 13374-13377 CC denotes and
T11750 13378-13382 JJ denotes long
T11752 13382-13383 HYPH denotes -
T11751 13383-13388 NN denotes range
T11753 13389-13392 NN denotes PCR
T11754 13392-13394 , denotes ,
T11736 13394-13400 NN denotes Figure
T11755 13401-13402 CD denotes 2
T11756 13402-13403 -RRB- denotes )
T11757 13403-13404 . denotes .
T11758 13404-13569 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 13405-13407 PRP denotes We
T11761 13408-13412 RB denotes next
T11760 13413-13419 VBD denotes tested
T11762 13420-13423 DT denotes the
T11763 13424-13434 NN denotes efficiency
T11764 13435-13437 IN denotes of
T11765 13438-13442 NN denotes RMCE
T11766 13443-13445 IN denotes in
T11767 13446-13448 NN denotes ES
T11768 13449-13454 NNS denotes cells
T11769 13454-13456 , denotes ,
T11770 13456-13461 VBG denotes using
T11771 13462-13463 DT denotes a
T11773 13464-13475 NN denotes replacement
T11772 13476-13485 NN denotes construct
T11774 13486-13494 VBG denotes encoding
T11775 13495-13498 NN denotes p53
T11776 13499-13504 VBN denotes fused
T11777 13505-13507 IN denotes to
T11778 13508-13511 NN denotes GFP
T11779 13512-13513 -LRB- denotes (
T11780 13513-13519 NN denotes p53GFP
T11781 13519-13520 -RRB- denotes )
T11782 13521-13523 TO denotes to
T11783 13524-13530 VB denotes enable
T11784 13531-13539 VBG denotes tracking
T11785 13540-13543 NN denotes p53
T11786 13544-13546 IN denotes in
T11787 13547-13557 JJ denotes individual
T11789 13558-13562 JJ denotes live
T11788 13563-13568 NNS denotes cells
T11790 13568-13569 . denotes .
T11791 13569-13736 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 13570-13581 RB denotes Importantly
T11794 13582-13589 RB denotes however
T11795 13589-13591 , denotes ,
T11796 13591-13594 NN denotes GFP
T11797 13595-13607 NN denotes fluorescence
T11798 13608-13611 VBD denotes was
T11799 13612-13615 RB denotes not
T11793 13616-13620 VBN denotes used
T11800 13621-13623 TO denotes to
T11801 13624-13630 VB denotes screen
T11802 13631-13636 NNS denotes cells
T11803 13637-13641 IN denotes with
T11804 13642-13650 VBN denotes targeted
T11805 13651-13657 NNS denotes events
T11806 13657-13659 , denotes ,
T11807 13659-13661 IN denotes as
T11809 13662-13664 PRP denotes we
T11808 13665-13671 VBD denotes wanted
T11810 13672-13674 TO denotes to
T11811 13675-13682 VB denotes develop
T11812 13683-13684 DT denotes a
T11814 13685-13692 JJ denotes general
T11813 13693-13699 NN denotes method
T11815 13700-13702 TO denotes to
T11816 13703-13710 VB denotes isolate
T11817 13711-13717 NN denotes marker
T11819 13717-13718 HYPH denotes -
T11818 13718-13722 JJ denotes free
T11820 13723-13735 NNS denotes recombinants
T11821 13735-13736 . denotes .
T11822 13736-13784 sentence denotes The exchange strategy is detailed in Figure 3A.
T11823 13737-13740 DT denotes The
T11825 13741-13749 NN denotes exchange
T11824 13750-13758 NN denotes strategy
T11827 13759-13761 VBZ denotes is
T11826 13762-13770 VBN denotes detailed
T11828 13771-13773 IN denotes in
T11829 13774-13780 NN denotes Figure
T11830 13781-13783 NN denotes 3A
T11831 13783-13784 . denotes .
T11832 13784-13955 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 13785-13787 PRP denotes We
T11834 13788-13794 VBD denotes picked
T11835 13795-13797 CD denotes 65
T11837 13798-13800 NN denotes ES
T11838 13801-13805 NN denotes cell
T11836 13806-13812 NNS denotes clones
T11839 13813-13822 JJ denotes resistant
T11840 13823-13825 IN denotes to
T11841 13826-13827 CD denotes 1
T11843 13827-13828 HYPH denotes -
T11844 13828-13829 -LRB- denotes (
T11846 13829-13830 HYPH denotes -
T11847 13830-13831 CD denotes 2
T11848 13831-13832 HYPH denotes -
T11849 13832-13837 NN denotes deoxy
T11850 13837-13838 HYPH denotes -
T11851 13838-13839 CD denotes 2
T11852 13839-13840 HYPH denotes -
T11853 13840-13846 NN denotes fluoro
T11854 13846-13847 HYPH denotes -
T11855 13847-13848 CD denotes 1
T11856 13848-13849 HYPH denotes -
T11857 13849-13850 NN denotes b
T11858 13850-13851 HYPH denotes -
T11859 13851-13852 NN denotes d
T11860 13852-13853 HYPH denotes -
T11861 13853-13860 NN denotes arabino
T11862 13860-13861 HYPH denotes -
T11845 13861-13870 NN denotes furanosyl
T11863 13870-13871 -RRB- denotes )
T11864 13871-13872 HYPH denotes -
T11842 13872-13873 CD denotes 5
T11866 13873-13874 HYPH denotes -
T11865 13874-13884 NN denotes iodouracil
T11867 13885-13886 -LRB- denotes (
T11868 13886-13890 NN denotes FIAU
T11869 13890-13891 -RRB- denotes )
T11870 13892-13895 IN denotes due
T11871 13896-13898 IN denotes to
T11872 13899-13901 NN denotes TK
T11873 13902-13906 NN denotes loss
T11874 13907-13910 CC denotes and
T11875 13911-13919 VBD denotes analyzed
T11876 13920-13925 PRP$ denotes their
T11877 13926-13929 NN denotes DNA
T11878 13930-13932 IN denotes by
T11879 13933-13936 NN denotes PCR
T11880 13937-13940 CC denotes and
T11881 13941-13949 NNP denotes Southern
T11882 13950-13954 NN denotes blot
T11883 13954-13955 . denotes .
T11884 13955-14051 sentence denotes Strikingly, 54 proper recombinants were identified, indicating very high RMCE efficiency (83%).
T11885 13956-13966 RB denotes Strikingly
T11887 13966-13968 , denotes ,
T11888 13968-13970 CD denotes 54
T11890 13971-13977 JJ denotes proper
T11889 13978-13990 NNS denotes recombinants
T11891 13991-13995 VBD denotes were
T11886 13996-14006 VBN denotes identified
T11892 14006-14008 , denotes ,
T11893 14008-14018 VBG denotes indicating
T11894 14019-14023 RB denotes very
T11895 14024-14028 JJ denotes high
T11897 14029-14033 NN denotes RMCE
T11896 14034-14044 NN denotes efficiency
T11898 14045-14046 -LRB- denotes (
T11900 14046-14048 CD denotes 83
T11899 14048-14049 NN denotes %
T11901 14049-14050 -RRB- denotes )
T11902 14050-14051 . denotes .
T11903 14051-14157 sentence denotes RMCE also proved to be precise, as no aberrant bands were detected in PCR and Southern blots (Figure 3A).
T11904 14052-14056 NN denotes RMCE
T11906 14057-14061 RB denotes also
T11905 14062-14068 VBD denotes proved
T11907 14069-14071 TO denotes to
T11908 14072-14074 VB denotes be
T11909 14075-14082 JJ denotes precise
T11910 14082-14084 , denotes ,
T11911 14084-14086 IN denotes as
T11913 14087-14089 DT denotes no
T11915 14090-14098 JJ denotes aberrant
T11914 14099-14104 NNS denotes bands
T11916 14105-14109 VBD denotes were
T11912 14110-14118 VBN denotes detected
T11917 14119-14121 IN denotes in
T11918 14122-14125 NN denotes PCR
T11919 14126-14129 CC denotes and
T11920 14130-14138 NNP denotes Southern
T11921 14139-14144 NNS denotes blots
T11922 14145-14146 -LRB- denotes (
T11924 14146-14152 NN denotes Figure
T11923 14153-14155 NN denotes 3A
T11925 14155-14156 -RRB- denotes )
T11926 14156-14157 . denotes .
T11927 14157-14311 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 14158-14161 NN denotes p53
T11930 14161-14162 SYM denotes +
T11931 14162-14163 HYPH denotes /
T11929 14163-14166 NN denotes GFP
T11933 14167-14169 NN denotes ES
T11932 14170-14176 NNS denotes clones
T11935 14177-14181 VBD denotes were
T11934 14182-14190 VBN denotes analyzed
T11936 14191-14193 IN denotes by
T11937 14194-14201 NNP denotes western
T11938 14202-14206 NN denotes blot
T11939 14207-14211 IN denotes with
T11940 14212-14214 DT denotes an
T11941 14215-14223 NN denotes antibody
T11942 14224-14231 IN denotes against
T11943 14232-14235 NN denotes p53
T11944 14235-14237 , denotes ,
T11945 14237-14240 CC denotes and
T11946 14241-14246 VBN denotes found
T11947 14247-14249 TO denotes to
T11948 14250-14257 VB denotes express
T11949 14258-14260 DT denotes an
T11951 14261-14271 JJ denotes additional
T11950 14272-14276 NN denotes band
T11952 14277-14279 IN denotes at
T11953 14280-14283 DT denotes the
T11955 14284-14292 VBN denotes expected
T11954 14293-14297 NN denotes size
T11956 14298-14299 -LRB- denotes (
T11957 14299-14302 NN denotes ca.
T11958 14303-14305 CD denotes 80
T11959 14306-14309 NN denotes kDa
T11960 14309-14310 -RRB- denotes )
T11961 14310-14311 . denotes .
T11962 14311-14635 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 14312-14324 RB denotes Surprisingly
T11965 14324-14326 , denotes ,
T11966 14326-14329 DT denotes the
T11967 14330-14336 NN denotes fusion
T11968 14337-14339 IN denotes of
T11969 14340-14343 NN denotes GFP
T11970 14344-14346 IN denotes to
T11971 14347-14350 NN denotes p53
T11972 14351-14361 RB denotes apparently
T11964 14362-14369 VBD denotes altered
T11974 14370-14373 NN denotes p53
T11975 14374-14383 NN denotes stability
T11976 14383-14385 : denotes :
T11977 14385-14391 JJ denotes steady
T11979 14391-14392 HYPH denotes -
T11978 14392-14397 NN denotes state
T11980 14398-14404 NNS denotes levels
T11981 14405-14407 IN denotes of
T11982 14408-14414 NN denotes p53GFP
T11973 14415-14419 VBD denotes were
T11983 14420-14424 RB denotes much
T11984 14425-14431 JJR denotes higher
T11985 14432-14436 IN denotes than
T11986 14437-14442 DT denotes those
T11987 14443-14445 IN denotes of
T11988 14446-14450 JJ denotes wild
T11990 14450-14451 HYPH denotes -
T11989 14451-14455 NN denotes type
T11991 14456-14459 NN denotes p53
T11992 14460-14461 -LRB- denotes (
T11993 14461-14466 NN denotes p53WT
T11994 14466-14467 -RRB- denotes )
T11995 14468-14470 IN denotes in
T11996 14471-14481 JJ denotes unstressed
T11997 14482-14487 NNS denotes cells
T11998 14487-14489 , denotes ,
T11999 14489-14492 CC denotes and
T12000 14493-14496 VBD denotes did
T12002 14497-14500 RB denotes not
T12001 14501-14505 VB denotes vary
T12003 14506-14519 RB denotes significantly
T12004 14520-14525 IN denotes after
T12005 14526-14529 NN denotes DNA
T12006 14530-14536 NN denotes damage
T12007 14536-14538 , denotes ,
T12008 14538-14540 IN denotes so
T12010 14541-14545 IN denotes that
T12011 14546-14549 DT denotes the
T12012 14550-14556 NNS denotes levels
T12013 14557-14560 IN denotes for
T12014 14561-14565 CC denotes both
T12015 14566-14571 NN denotes p53WT
T12016 14572-14575 CC denotes and
T12017 14576-14582 NN denotes p53GFP
T12009 14583-14587 VBD denotes were
T12018 14588-14595 JJ denotes similar
T12019 14596-14601 IN denotes after
T12020 14602-14612 NN denotes adriamycin
T12021 14613-14622 NN denotes treatment
T12022 14623-14624 -LRB- denotes (
T12024 14624-14630 NN denotes Figure
T12023 14631-14633 NN denotes 3A
T12025 14633-14634 -RRB- denotes )
T12026 14634-14635 . denotes .
T12027 14635-14768 sentence denotes Six independent p53+/GFP clones were injected into blastocysts and transferred to pseudo-pregnant females using standard procedures.
T12028 14636-14639 CD denotes Six
T12030 14640-14651 JJ denotes independent
T12031 14652-14655 NN denotes p53
T12033 14655-14656 SYM denotes +
T12034 14656-14657 HYPH denotes /
T12032 14657-14660 NN denotes GFP
T12029 14661-14667 NNS denotes clones
T12036 14668-14672 VBD denotes were
T12035 14673-14681 VBN denotes injected
T12037 14682-14686 IN denotes into
T12038 14687-14698 NNS denotes blastocysts
T12039 14699-14702 CC denotes and
T12040 14703-14714 VBN denotes transferred
T12041 14715-14717 IN denotes to
T12042 14718-14724 JJ denotes pseudo
T12044 14724-14725 HYPH denotes -
T12043 14725-14733 JJ denotes pregnant
T12045 14734-14741 NNS denotes females
T12046 14742-14747 VBG denotes using
T12047 14748-14756 JJ denotes standard
T12048 14757-14767 NNS denotes procedures
T12049 14767-14768 . denotes .
T12050 14768-14810 sentence denotes Strikingly, no pregnancies were obtained.
T12051 14769-14779 RB denotes Strikingly
T12053 14779-14781 , denotes ,
T12054 14781-14783 DT denotes no
T12055 14784-14795 NNS denotes pregnancies
T12056 14796-14800 VBD denotes were
T12052 14801-14809 VBN denotes obtained
T12057 14809-14810 . denotes .
T12058 14810-15024 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 14811-14813 PRP denotes It
T12061 14814-14817 VBZ denotes has
T12062 14818-14822 VBN denotes been
T12060 14823-14828 VBN denotes shown
T12064 14829-14833 IN denotes that
T12066 14834-14837 DT denotes the
T12068 14838-14841 NN denotes p53
T12067 14842-14849 NN denotes pathway
T12069 14850-14852 VBZ denotes is
T12065 14853-14862 VBN denotes regulated
T12070 14863-14867 RB denotes very
T12071 14868-14879 RB denotes differently
T12072 14880-14882 IN denotes in
T12073 14883-14885 NN denotes ES
T12075 14886-14889 CC denotes and
T12076 14890-14897 JJ denotes somatic
T12074 14898-14903 NNS denotes cells
T12077 14903-14905 : denotes :
T12078 14905-14907 NN denotes ES
T12079 14908-14913 NNS denotes cells
T12063 14914-14921 VBP denotes contain
T12080 14922-14932 RB denotes relatively
T12081 14933-14937 JJ denotes high
T12083 14938-14941 NN denotes p53
T12082 14942-14948 NNS denotes levels
T12084 14949-14952 CC denotes and
T12085 14953-14957 VBP denotes lack
T12086 14958-14961 DT denotes the
T12088 14962-14965 NN denotes p53
T12089 14965-14966 HYPH denotes -
T12090 14966-14974 JJ denotes mediated
T12091 14975-14978 NN denotes DNA
T12092 14979-14985 NN denotes damage
T12087 14986-14995 NNS denotes responses
T12093 14996-15001 VBN denotes found
T12094 15002-15004 IN denotes in
T12095 15005-15012 JJ denotes somatic
T12096 15013-15018 NNS denotes cells
T12097 15019-15020 -LRB- denotes (
T12098 15020-15022 CD denotes 18
T12099 15022-15023 -RRB- denotes )
T12100 15023-15024 . denotes .
T12101 15024-15396 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 15025-15029 DT denotes This
T12104 15029-15031 , denotes ,
T12105 15031-15039 RB denotes together
T12106 15040-15044 IN denotes with
T12107 15045-15048 DT denotes the
T12108 15049-15060 NN denotes observation
T12109 15061-15065 IN denotes that
T12111 15066-15069 NN denotes p53
T12112 15070-15076 NNS denotes levels
T12110 15077-15085 VBP denotes decrease
T12113 15086-15092 IN denotes during
T12114 15093-15098 NN denotes mouse
T12115 15099-15112 NN denotes embryogenesis
T12116 15113-15114 -LRB- denotes (
T12117 15114-15116 CD denotes 19
T12118 15116-15117 -RRB- denotes )
T12119 15117-15119 , denotes ,
T12103 15119-15128 VBD denotes suggested
T12121 15129-15131 DT denotes an
T12122 15132-15143 NN denotes explanation
T12123 15144-15147 IN denotes for
T12124 15148-15151 DT denotes the
T12126 15152-15160 VBN denotes observed
T12125 15161-15165 NN denotes lack
T12127 15166-15168 IN denotes of
T12128 15169-15180 NNS denotes pregnancies
T12129 15180-15182 : denotes :
T12130 15182-15184 PRP denotes we
T12120 15185-15194 VBP denotes speculate
T12131 15195-15199 IN denotes that
T12133 15200-15203 DT denotes the
T12135 15204-15208 JJ denotes high
T12134 15209-15215 NNS denotes levels
T12136 15216-15218 IN denotes of
T12137 15219-15225 NN denotes p53GFP
T12138 15226-15228 IN denotes in
T12139 15229-15232 DT denotes the
T12141 15233-15235 NN denotes ES
T12140 15236-15241 NNS denotes cells
T12142 15242-15250 VBN denotes injected
T12143 15251-15255 IN denotes into
T12144 15256-15267 NNS denotes blastocysts
T12145 15268-15273 MD denotes might
T12146 15274-15278 VB denotes have
T12132 15279-15288 VBN denotes prevented
T12147 15289-15295 JJ denotes normal
T12149 15296-15305 JJ denotes embryonic
T12148 15306-15317 NN denotes development
T12150 15318-15322 IN denotes once
T12152 15323-15328 DT denotes these
T12153 15329-15334 NNS denotes cells
T12151 15335-15340 VBD denotes began
T12154 15341-15343 TO denotes to
T12155 15344-15357 VB denotes differentiate
T12156 15358-15361 CC denotes and
T12157 15362-15365 DT denotes the
T12159 15366-15369 NN denotes p53
T12158 15370-15377 NN denotes pathway
T12160 15378-15384 VBD denotes became
T12161 15385-15395 JJ denotes functional
T12162 15395-15396 . denotes .
T12163 15396-15559 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 15397-15399 TO denotes To
T12165 15400-15404 VB denotes test
T12167 15405-15409 DT denotes this
T12168 15410-15421 NN denotes possibility
T12169 15421-15423 , denotes ,
T12170 15423-15425 PRP denotes we
T12166 15426-15435 VBD denotes performed
T12171 15436-15440 NN denotes RMCE
T12172 15441-15445 IN denotes with
T12173 15446-15447 DT denotes a
T12175 15448-15451 NN denotes p53
T12176 15452-15458 NN denotes fusion
T12174 15459-15463 NN denotes gene
T12177 15464-15466 IN denotes in
T12179 15467-15472 WDT denotes which
T12180 15473-15476 DT denotes the
T12182 15477-15480 NN denotes p53
T12183 15481-15488 NN denotes proline
T12185 15488-15489 HYPH denotes -
T12184 15489-15493 JJ denotes rich
T12181 15494-15500 NN denotes domain
T12186 15501-15502 -LRB- denotes (
T12187 15502-15505 NN denotes PRD
T12188 15505-15506 -RRB- denotes )
T12189 15507-15510 VBD denotes was
T12178 15511-15518 VBN denotes deleted
T12190 15519-15520 -LRB- denotes (
T12191 15520-15525 NN denotes p53ΔP
T12192 15525-15526 -RRB- denotes )
T12193 15526-15528 , denotes ,
T12194 15528-15531 CC denotes and
T12195 15532-15535 DT denotes the
T12196 15536-15541 NN denotes p53ΔP
T12198 15542-15545 VBD denotes was
T12197 15546-15551 VBN denotes fused
T12199 15552-15554 IN denotes to
T12200 15555-15558 NN denotes GFP
T12201 15558-15559 . denotes .
T12202 15559-15690 sentence denotes We used this mutant because deleting the proline-rich domain decreases stability and compromises DNA-damage responses in vivo (5).
T12203 15560-15562 PRP denotes We
T12204 15563-15567 VBD denotes used
T12205 15568-15572 DT denotes this
T12206 15573-15579 NN denotes mutant
T12207 15580-15587 IN denotes because
T12209 15588-15596 VBG denotes deleting
T12210 15597-15600 DT denotes the
T12212 15601-15608 NN denotes proline
T12214 15608-15609 HYPH denotes -
T12213 15609-15613 JJ denotes rich
T12211 15614-15620 NN denotes domain
T12208 15621-15630 VBZ denotes decreases
T12215 15631-15640 NN denotes stability
T12216 15641-15644 CC denotes and
T12217 15645-15656 VBZ denotes compromises
T12218 15657-15660 NN denotes DNA
T12220 15660-15661 HYPH denotes -
T12219 15661-15667 NN denotes damage
T12221 15668-15677 NNS denotes responses
T12222 15678-15680 FW denotes in
T12223 15681-15685 FW denotes vivo
T12224 15686-15687 -LRB- denotes (
T12225 15687-15688 CD denotes 5
T12226 15688-15689 -RRB- denotes )
T12227 15689-15690 . denotes .
T12228 15690-15785 sentence denotes According to our hypothesis, this hypomorphic mutant should not prevent embryonic development.
T12229 15691-15700 VBG denotes According
T12231 15701-15703 IN denotes to
T12232 15704-15707 PRP$ denotes our
T12233 15708-15718 NN denotes hypothesis
T12234 15718-15720 , denotes ,
T12235 15720-15724 DT denotes this
T12237 15725-15736 JJ denotes hypomorphic
T12236 15737-15743 NN denotes mutant
T12238 15744-15750 MD denotes should
T12239 15751-15754 RB denotes not
T12230 15755-15762 VB denotes prevent
T12240 15763-15772 JJ denotes embryonic
T12241 15773-15784 NN denotes development
T12242 15784-15785 . denotes .
T12243 15785-15972 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 15786-15790 NN denotes RMCE
T12246 15791-15795 IN denotes with
T12247 15796-15797 DT denotes a
T12249 15798-15806 NN denotes p53ΔPGFP
T12250 15807-15818 NN denotes replacement
T12248 15819-15826 NN denotes plasmid
T12245 15827-15830 VBD denotes was
T12251 15831-15836 RB denotes again
T12252 15837-15841 RB denotes very
T12253 15842-15851 JJ denotes efficient
T12254 15852-15855 CC denotes and
T12255 15856-15863 NNP denotes western
T12256 15864-15869 NNS denotes blots
T12257 15870-15878 VBD denotes revealed
T12258 15879-15881 DT denotes an
T12260 15882-15892 JJ denotes additional
T12259 15893-15897 NN denotes band
T12261 15898-15900 IN denotes of
T12262 15901-15904 DT denotes the
T12264 15905-15914 VBN denotes predicted
T12265 15915-15924 JJ denotes molecular
T12263 15925-15931 NN denotes weight
T12266 15932-15936 RB denotes only
T12267 15937-15939 IN denotes in
T12268 15940-15943 NN denotes p53
T12270 15943-15944 SYM denotes +
T12271 15944-15945 HYPH denotes /
T12269 15945-15950 NN denotes ΔPGFP
T12273 15951-15953 NN denotes ES
T12272 15954-15959 NNS denotes cells
T12274 15960-15961 -LRB- denotes (
T12276 15961-15967 NN denotes Figure
T12275 15968-15970 NN denotes 3B
T12277 15970-15971 -RRB- denotes )
T12278 15971-15972 . denotes .
T12279 15972-16124 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 15973-15975 IN denotes As
T12281 15976-15984 VBN denotes expected
T12283 15984-15986 , denotes ,
T12284 15986-15989 DT denotes the
T12286 15990-15993 NN denotes PRD
T12285 15994-16002 NN denotes deletion
T12282 16003-16013 VBD denotes correlated
T12288 16014-16018 IN denotes with
T12289 16019-16024 JJR denotes lower
T12291 16025-16035 NN denotes expression
T12290 16036-16042 NNS denotes levels
T12292 16042-16044 : denotes :
T12293 16044-16052 NN denotes p53ΔPGFP
T12287 16053-16056 VBD denotes was
T12294 16057-16061 RB denotes much
T12295 16062-16066 RBR denotes less
T12296 16067-16075 JJ denotes abundant
T12297 16076-16080 IN denotes than
T12298 16081-16086 NN denotes p53WT
T12299 16087-16089 IN denotes in
T12300 16090-16093 DT denotes all
T12302 16094-16097 NN denotes p53
T12304 16097-16098 SYM denotes +
T12305 16098-16099 HYPH denotes /
T12303 16099-16104 NN denotes ΔPGFP
T12301 16105-16111 NNS denotes clones
T12306 16112-16113 -LRB- denotes (
T12308 16113-16119 NN denotes Figure
T12307 16120-16122 NN denotes 3B
T12309 16122-16123 -RRB- denotes )
T12310 16123-16124 . denotes .
T12311 16124-16255 sentence denotes We next determined whether p53+/ΔPGFP ES cells could generate chimeric mice and transmit the modified allele through the germline.
T12312 16125-16127 PRP denotes We
T12314 16128-16132 RB denotes next
T12313 16133-16143 VBD denotes determined
T12315 16144-16151 IN denotes whether
T12317 16152-16155 NN denotes p53
T12319 16155-16156 SYM denotes +
T12320 16156-16157 HYPH denotes /
T12318 16157-16162 NN denotes ΔPGFP
T12322 16163-16165 NN denotes ES
T12321 16166-16171 NNS denotes cells
T12323 16172-16177 MD denotes could
T12316 16178-16186 VB denotes generate
T12324 16187-16195 JJ denotes chimeric
T12325 16196-16200 NNS denotes mice
T12326 16201-16204 CC denotes and
T12327 16205-16213 VB denotes transmit
T12328 16214-16217 DT denotes the
T12330 16218-16226 VBN denotes modified
T12329 16227-16233 NN denotes allele
T12331 16234-16241 IN denotes through
T12332 16242-16245 DT denotes the
T12333 16246-16254 NN denotes germline
T12334 16254-16255 . denotes .
T12335 16255-16363 sentence denotes Two p53+/ΔPGFP ES cell clones were injected into blastocysts and highly chimeric (>80%) mice were obtained.
T12336 16256-16259 CD denotes Two
T12338 16260-16263 NN denotes p53
T12340 16263-16264 SYM denotes +
T12341 16264-16265 HYPH denotes /
T12339 16265-16270 NN denotes ΔPGFP
T12342 16271-16273 NN denotes ES
T12343 16274-16278 NN denotes cell
T12337 16279-16285 NNS denotes clones
T12345 16286-16290 VBD denotes were
T12344 16291-16299 VBN denotes injected
T12346 16300-16304 IN denotes into
T12347 16305-16316 NNS denotes blastocysts
T12348 16317-16320 CC denotes and
T12349 16321-16327 RB denotes highly
T12350 16328-16336 JJ denotes chimeric
T12352 16337-16338 -LRB- denotes (
T12353 16338-16339 SYM denotes >
T12354 16339-16341 CD denotes 80
T12351 16341-16342 NN denotes %
T12356 16342-16343 -RRB- denotes )
T12355 16344-16348 NNS denotes mice
T12358 16349-16353 VBD denotes were
T12357 16354-16362 VBN denotes obtained
T12359 16362-16363 . denotes .
T12360 16363-16451 sentence denotes Heterozygote pups were recovered from the mating the chimeras with WT mice (Figure 3C).
T12361 16364-16376 NN denotes Heterozygote
T12362 16377-16381 NNS denotes pups
T12364 16382-16386 VBD denotes were
T12363 16387-16396 VBN denotes recovered
T12365 16397-16401 IN denotes from
T12366 16402-16405 DT denotes the
T12367 16406-16412 NN denotes mating
T12368 16413-16416 DT denotes the
T12369 16417-16425 NNS denotes chimeras
T12370 16426-16430 IN denotes with
T12371 16431-16433 NN denotes WT
T12372 16434-16438 NNS denotes mice
T12373 16439-16440 -LRB- denotes (
T12375 16440-16446 NN denotes Figure
T12374 16447-16449 NN denotes 3C
T12376 16449-16450 -RRB- denotes )
T12377 16450-16451 . denotes .
T12378 16451-16606 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 16452-16457 DT denotes These
T12380 16458-16462 NNS denotes data
T12381 16463-16474 VBP denotes demonstrate
T12382 16475-16479 IN denotes that
T12384 16480-16486 NN denotes marker
T12386 16486-16487 HYPH denotes -
T12385 16487-16491 JJ denotes free
T12387 16492-16496 NN denotes RMCE
T12383 16497-16499 VBZ denotes is
T12388 16500-16504 RB denotes very
T12389 16505-16514 JJ denotes efficient
T12390 16515-16517 IN denotes in
T12391 16518-16520 NN denotes ES
T12392 16521-16526 NNS denotes cells
T12393 16527-16530 CC denotes and
T12394 16531-16537 VBZ denotes allows
T12395 16538-16546 NN denotes germline
T12396 16547-16559 NN denotes transmission
T12397 16560-16562 IN denotes of
T12398 16563-16564 DT denotes a
T12400 16565-16573 VBN denotes targeted
T12399 16574-16582 NN denotes mutation
T12401 16583-16584 -LRB- denotes (
T12402 16584-16587 VB denotes see
T12403 16588-16594 NN denotes Figure
T12404 16595-16596 CD denotes 1
T12405 16596-16598 , denotes ,
T12406 16598-16602 NN denotes path
T12407 16603-16604 NN denotes A
T12408 16604-16605 -RRB- denotes )
T12409 16605-16606 . denotes .
T12410 16606-16740 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 16607-16609 PRP denotes We
T12413 16610-16614 RB denotes next
T12412 16615-16625 VBD denotes determined
T12414 16626-16633 IN denotes whether
T12416 16634-16637 DT denotes the
T12418 16638-16642 NN denotes RMCE
T12417 16643-16651 NN denotes approach
T12419 16652-16657 MD denotes could
T12420 16658-16660 VB denotes be
T12415 16661-16665 VBN denotes used
T12421 16666-16668 TO denotes to
T12422 16669-16675 VB denotes target
T12423 16676-16685 NNS denotes mutations
T12424 16686-16688 IN denotes at
T12425 16689-16692 DT denotes the
T12427 16693-16696 NN denotes p53
T12426 16697-16703 NN denotes allele
T12428 16704-16706 IN denotes in
T12429 16707-16714 JJ denotes somatic
T12430 16715-16720 NNS denotes cells
T12431 16721-16722 -LRB- denotes (
T12432 16722-16728 NN denotes Figure
T12433 16729-16730 CD denotes 1
T12434 16730-16732 , denotes ,
T12435 16732-16736 NN denotes path
T12436 16737-16738 NN denotes B
T12437 16738-16739 -RRB- denotes )
T12438 16739-16740 . denotes .
T12439 16740-16903 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 16741-16743 PRP denotes We
T12442 16744-16749 RB denotes first
T12441 16750-16758 VBD denotes verified
T12443 16759-16763 IN denotes that
T12445 16764-16767 DT denotes the
T12447 16768-16772 NN denotes RMCE
T12449 16772-16773 HYPH denotes -
T12448 16773-16778 JJ denotes ready
T12450 16779-16782 NN denotes p53
T12446 16783-16788 NN denotes locus
T12451 16789-16790 -LRB- denotes (
T12452 16790-16797 NN denotes p53RMCE
T12453 16797-16798 -RRB- denotes )
T12454 16799-16804 MD denotes could
T12455 16805-16807 VB denotes be
T12444 16808-16819 VBN denotes transmitted
T12456 16820-16827 IN denotes through
T12457 16828-16831 DT denotes the
T12458 16832-16840 NN denotes germline
T12459 16841-16843 IN denotes by
T12460 16844-16850 VBG denotes mating
T12461 16851-16858 NN denotes p53RMCE
T12463 16858-16859 HYPH denotes /
T12464 16859-16860 SYM denotes +
T12462 16861-16869 NNS denotes chimeras
T12465 16870-16874 IN denotes with
T12466 16875-16878 NN denotes p53
T12468 16878-16879 SYM denotes +
T12469 16879-16880 HYPH denotes /
T12470 16880-16881 SYM denotes −
T12467 16882-16886 NNS denotes mice
T12471 16887-16888 -LRB- denotes (
T12472 16888-16890 CD denotes 20
T12473 16890-16891 -RRB- denotes )
T12474 16892-16893 -LRB- denotes (
T12475 16893-16899 NN denotes Figure
T12476 16900-16901 CD denotes 4
T12477 16901-16902 -RRB- denotes )
T12478 16902-16903 . denotes .
T12479 16903-17024 sentence denotes Importantly, this allowed us to generate p53RMCE/− MEFs, which were used to test RMCE at the p53 locus in somatic cells.
T12480 16904-16915 RB denotes Importantly
T12482 16915-16917 , denotes ,
T12483 16917-16921 DT denotes this
T12481 16922-16929 VBD denotes allowed
T12484 16930-16932 PRP denotes us
T12486 16933-16935 TO denotes to
T12485 16936-16944 VB denotes generate
T12487 16945-16952 NN denotes p53RMCE
T12489 16952-16953 HYPH denotes /
T12490 16953-16954 SYM denotes −
T12488 16955-16959 NNS denotes MEFs
T12491 16959-16961 , denotes ,
T12492 16961-16966 WDT denotes which
T12494 16967-16971 VBD denotes were
T12493 16972-16976 VBN denotes used
T12495 16977-16979 TO denotes to
T12496 16980-16984 VB denotes test
T12497 16985-16989 NN denotes RMCE
T12498 16990-16992 IN denotes at
T12499 16993-16996 DT denotes the
T12501 16997-17000 NN denotes p53
T12500 17001-17006 NN denotes locus
T12502 17007-17009 IN denotes in
T12503 17010-17017 JJ denotes somatic
T12504 17018-17023 NNS denotes cells
T12505 17023-17024 . denotes .
T12506 17024-17192 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 17025-17027 PRP denotes We
T12509 17028-17033 RB denotes first
T12508 17034-17043 VBD denotes attempted
T12510 17044-17048 NN denotes RMCE
T12511 17049-17051 IN denotes in
T12512 17052-17056 NNS denotes MEFs
T12513 17057-17059 IN denotes by
T12514 17060-17075 VBG denotes electroporating
T12515 17076-17083 NN denotes p53RMCE
T12517 17083-17084 HYPH denotes /
T12518 17084-17085 SYM denotes −
T12516 17086-17090 NNS denotes MEFs
T12519 17091-17095 IN denotes with
T12520 17096-17097 DT denotes a
T12522 17098-17101 NN denotes Cre
T12524 17101-17102 HYPH denotes -
T12523 17102-17112 NN denotes expression
T12521 17113-17120 NN denotes plasmid
T12525 17121-17124 CC denotes and
T12526 17125-17128 DT denotes the
T12528 17129-17135 NN denotes p53GFP
T12527 17136-17143 NN denotes plasmid
T12529 17143-17145 , denotes ,
T12530 17145-17153 VBN denotes followed
T12531 17154-17156 IN denotes by
T12532 17157-17166 NN denotes selection
T12533 17167-17171 IN denotes with
T12534 17172-17176 NN denotes FIAU
T12535 17177-17179 CC denotes or
T12536 17180-17191 NN denotes ganciclovir
T12537 17191-17192 . denotes .
T12538 17192-17273 sentence denotes Strikingly, no clones with an exchanged allele were identified (data not shown).
T12539 17193-17203 RB denotes Strikingly
T12541 17203-17205 , denotes ,
T12542 17205-17207 DT denotes no
T12543 17208-17214 NNS denotes clones
T12544 17215-17219 IN denotes with
T12545 17220-17222 DT denotes an
T12547 17223-17232 VBN denotes exchanged
T12546 17233-17239 NN denotes allele
T12548 17240-17244 VBD denotes were
T12540 17245-17255 VBN denotes identified
T12549 17256-17257 -LRB- denotes (
T12551 17257-17261 NNS denotes data
T12552 17262-17265 RB denotes not
T12550 17266-17271 VBN denotes shown
T12553 17271-17272 -RRB- denotes )
T12554 17272-17273 . denotes .
T12555 17273-17476 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 17274-17278 NN denotes RMCE
T12558 17279-17283 IN denotes with
T12559 17284-17290 NN denotes p53GFP
T12560 17291-17293 IN denotes in
T12561 17294-17296 NN denotes ES
T12562 17297-17302 NNS denotes cells
T12557 17303-17309 VBD denotes showed
T12563 17310-17314 IN denotes that
T12565 17315-17321 NN denotes p53GFP
T12566 17322-17324 VBZ denotes is
T12564 17325-17334 VBN denotes expressed
T12567 17335-17337 IN denotes at
T12568 17338-17342 JJ denotes high
T12569 17343-17349 NNS denotes levels
T12570 17350-17351 -LRB- denotes (
T12572 17351-17357 NN denotes Figure
T12571 17358-17360 NN denotes 3A
T12573 17360-17361 -RRB- denotes )
T12574 17361-17363 , denotes ,
T12575 17363-17366 CC denotes and
T12576 17367-17369 IN denotes as
T12577 17370-17379 VBN denotes mentioned
T12579 17380-17386 RB denotes before
T12580 17386-17388 , denotes ,
T12581 17388-17391 DT denotes the
T12583 17392-17395 NN denotes p53
T12582 17396-17403 NN denotes pathway
T12584 17404-17408 WDT denotes that
T12586 17409-17412 MD denotes can
T12587 17413-17415 VB denotes be
T12585 17416-17425 VBN denotes activated
T12588 17426-17428 IN denotes in
T12589 17429-17433 NNS denotes MEFs
T12590 17434-17436 VBZ denotes is
T12591 17437-17440 RB denotes not
T12592 17441-17448 RB denotes readily
T12578 17449-17458 VBN denotes activated
T12593 17459-17461 IN denotes in
T12594 17462-17464 NN denotes ES
T12595 17465-17470 NNS denotes cells
T12596 17471-17472 -LRB- denotes (
T12597 17472-17474 CD denotes 18
T12598 17474-17475 -RRB- denotes )
T12599 17475-17476 . denotes .
T12600 17476-17645 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 17477-17480 DT denotes The
T12602 17481-17488 NNS denotes results
T12604 17489-17494 RB denotes above
T12603 17495-17502 VBP denotes suggest
T12605 17503-17507 IN denotes that
T12607 17508-17512 JJ denotes high
T12608 17513-17519 NNS denotes levels
T12609 17520-17522 IN denotes of
T12610 17523-17529 NN denotes p53GFP
T12611 17530-17535 MD denotes could
T12606 17536-17538 VB denotes be
T12612 17539-17548 VBN denotes tolerated
T12613 17549-17551 IN denotes by
T12614 17552-17554 NN denotes ES
T12615 17555-17560 NNS denotes cells
T12616 17561-17564 CC denotes but
T12617 17565-17570 JJ denotes toxic
T12618 17571-17573 IN denotes to
T12619 17574-17578 NNS denotes MEFs
T12620 17578-17580 , denotes ,
T12621 17580-17582 IN denotes so
T12623 17583-17587 IN denotes that
T12624 17588-17592 NNS denotes MEFs
T12625 17593-17595 IN denotes in
T12627 17596-17601 WDT denotes which
T12628 17602-17604 DT denotes an
T12629 17605-17609 NN denotes RMCE
T12630 17610-17613 VBD denotes had
T12626 17614-17622 VBN denotes occurred
T12622 17623-17629 VBD denotes failed
T12631 17630-17632 TO denotes to
T12632 17633-17644 VB denotes proliferate
T12633 17644-17645 . denotes .
T12634 17645-17855 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 17646-17648 TO denotes To
T12636 17649-17653 VB denotes test
T12638 17654-17658 DT denotes this
T12639 17659-17670 NN denotes possibility
T12640 17670-17672 , denotes ,
T12641 17672-17679 NN denotes p53RMCE
T12643 17679-17680 HYPH denotes /
T12644 17680-17681 SYM denotes −
T12642 17682-17686 NNS denotes MEFs
T12645 17687-17691 VBD denotes were
T12637 17692-17706 VBN denotes electroporated
T12646 17707-17711 IN denotes with
T12647 17712-17715 DT denotes the
T12649 17716-17722 NN denotes p53GFP
T12650 17723-17734 NN denotes replacement
T12648 17735-17744 NN denotes construct
T12651 17745-17749 IN denotes with
T12652 17750-17752 CC denotes or
T12653 17753-17760 IN denotes without
T12654 17761-17762 DT denotes a
T12656 17763-17766 NN denotes Cre
T12658 17766-17767 HYPH denotes -
T12657 17767-17777 NN denotes expression
T12655 17778-17785 NN denotes plasmid
T12659 17785-17787 , denotes ,
T12660 17787-17791 RB denotes then
T12661 17792-17800 VBN denotes analyzed
T12662 17801-17803 IN denotes by
T12663 17804-17816 NN denotes fluorescence
T12664 17817-17827 NN denotes microscopy
T12665 17828-17830 CD denotes 48
T12666 17831-17832 NN denotes h
T12667 17833-17838 IN denotes after
T12668 17839-17854 NN denotes electroporation
T12669 17854-17855 . denotes .
T12670 17855-18073 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 17856-17859 DT denotes The
T12672 17860-17870 NN denotes experiment
T12674 17871-17874 VBD denotes was
T12673 17875-17879 VBN denotes done
T12675 17880-17887 IN denotes without
T12676 17888-17897 NN denotes selection
T12677 17898-17900 TO denotes to
T12678 17901-17907 VB denotes enable
T12679 17908-17919 NN denotes observation
T12680 17920-17922 IN denotes of
T12681 17923-17928 NNS denotes cells
T12682 17929-17934 IN denotes under
T12683 17935-17945 NNS denotes conditions
T12684 17946-17951 WRB denotes where
T12686 17952-17953 DT denotes a
T12687 17954-17961 NN denotes failure
T12688 17962-17964 TO denotes to
T12689 17965-17976 VB denotes proliferate
T12690 17977-17982 MD denotes would
T12691 17983-17986 RB denotes not
T12685 17987-17993 VB denotes derive
T12692 17994-17998 IN denotes from
T12693 17999-18003 NN denotes FIAU
T12695 18004-18006 CC denotes or
T12696 18007-18018 NN denotes ganciclovir
T12694 18019-18027 NN denotes toxicity
T12697 18028-18031 CC denotes but
T12698 18032-18038 RB denotes rather
T12699 18039-18045 RB denotes solely
T12700 18046-18050 IN denotes from
T12701 18051-18054 DT denotes the
T12702 18055-18062 NNS denotes effects
T12703 18063-18065 IN denotes of
T12704 18066-18072 NN denotes p53GFP
T12705 18072-18073 . denotes .
T12706 18073-18216 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 18074-18076 PRP denotes We
T12708 18077-18085 VBD denotes observed
T12709 18086-18087 DT denotes a
T12711 18088-18091 JJ denotes few
T12712 18092-18103 JJ denotes fluorescent
T12710 18104-18109 NNS denotes cells
T12713 18110-18114 RB denotes only
T12714 18115-18119 WRB denotes when
T12716 18120-18123 DT denotes the
T12718 18124-18127 NN denotes Cre
T12719 18128-18138 NN denotes expression
T12717 18139-18146 NN denotes plasmid
T12720 18147-18150 VBD denotes was
T12715 18151-18168 VBN denotes co-electroporated
T12721 18168-18170 , denotes ,
T12722 18170-18180 VBG denotes suggesting
T12723 18181-18185 IN denotes that
T12725 18186-18190 JJ denotes such
T12726 18191-18196 NNS denotes cells
T12724 18197-18205 VBD denotes resulted
T12727 18206-18210 IN denotes from
T12728 18211-18215 NN denotes RMCE
T12729 18215-18216 . denotes .
T12730 18216-18399 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 18217-18228 RB denotes Importantly
T12733 18228-18230 , denotes ,
T12734 18230-18233 DT denotes the
T12736 18234-18238 JJ denotes rare
T12737 18239-18250 JJ denotes fluorescent
T12735 18251-18256 NNS denotes cells
T12732 18257-18260 VBD denotes had
T12738 18261-18262 DT denotes a
T12740 18263-18267 JJ denotes flat
T12741 18267-18269 , denotes ,
T12742 18269-18270 `` denotes ‘
T12743 18270-18275 JJ denotes fried
T12745 18275-18276 HYPH denotes -
T12744 18276-18279 NN denotes egg
T12746 18279-18280 '' denotes ’
T12739 18281-18291 NN denotes appearance
T12747 18292-18299 JJ denotes typical
T12748 18300-18302 IN denotes of
T12749 18303-18312 JJ denotes senescent
T12750 18313-18318 NNS denotes cells
T12751 18319-18320 -LRB- denotes (
T12753 18320-18326 NN denotes Figure
T12752 18327-18329 NN denotes 5A
T12754 18329-18330 -RRB- denotes )
T12755 18330-18332 , denotes ,
T12756 18332-18335 CC denotes and
T12757 18336-18340 WRB denotes when
T12759 18341-18347 NNS denotes plates
T12760 18348-18352 VBD denotes were
T12758 18353-18361 VBN denotes observed
T12762 18362-18363 CD denotes 5
T12763 18364-18368 NNS denotes days
T12764 18369-18374 RB denotes later
T12765 18374-18376 , denotes ,
T12766 18376-18379 DT denotes the
T12767 18380-18385 NNS denotes cells
T12768 18386-18389 VBD denotes had
T12761 18390-18398 VBN denotes detached
T12769 18398-18399 . denotes .
T12770 18399-18529 sentence denotes Altogether, the results suggest that RMCE can give rise to p53GFP-expressing MEFs, but they die rapidly owing to p53GFP toxicity.
T12771 18400-18410 RB denotes Altogether
T12773 18410-18412 , denotes ,
T12774 18412-18415 DT denotes the
T12775 18416-18423 NNS denotes results
T12772 18424-18431 VBP denotes suggest
T12776 18432-18436 IN denotes that
T12778 18437-18441 NN denotes RMCE
T12779 18442-18445 MD denotes can
T12777 18446-18450 VB denotes give
T12780 18451-18455 NN denotes rise
T12781 18456-18458 IN denotes to
T12782 18459-18465 NN denotes p53GFP
T12784 18465-18466 HYPH denotes -
T12783 18466-18476 VBG denotes expressing
T12785 18477-18481 NNS denotes MEFs
T12786 18481-18483 , denotes ,
T12787 18483-18486 CC denotes but
T12788 18487-18491 PRP denotes they
T12789 18492-18495 VBP denotes die
T12790 18496-18503 RB denotes rapidly
T12791 18504-18509 VBG denotes owing
T12792 18510-18512 IN denotes to
T12793 18513-18519 NN denotes p53GFP
T12794 18520-18528 NN denotes toxicity
T12795 18528-18529 . denotes .
T12796 18529-18599 sentence denotes We also performed RMCE in p53RMCE/− MEFs with the p53ΔPGFP construct.
T12797 18530-18532 PRP denotes We
T12799 18533-18537 RB denotes also
T12798 18538-18547 VBD denotes performed
T12800 18548-18552 NN denotes RMCE
T12801 18553-18555 IN denotes in
T12802 18556-18563 NN denotes p53RMCE
T12804 18563-18564 HYPH denotes /
T12805 18564-18565 SYM denotes −
T12803 18566-18570 NNS denotes MEFs
T12806 18571-18575 IN denotes with
T12807 18576-18579 DT denotes the
T12809 18580-18588 NN denotes p53ΔPGFP
T12808 18589-18598 NN denotes construct
T12810 18598-18599 . denotes .
T12811 18599-18792 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 18600-18608 NN denotes p53ΔPGFP
T12814 18608-18609 HYPH denotes -
T12813 18609-18619 VBG denotes expressing
T12815 18620-18624 NNS denotes MEFs
T12816 18625-18629 VBD denotes were
T12817 18630-18636 JJ denotes viable
T12818 18636-18638 , denotes ,
T12819 18638-18647 VBD denotes recovered
T12820 18648-18652 IN denotes with
T12821 18653-18655 DT denotes an
T12822 18656-18666 NN denotes efficiency
T12823 18667-18669 IN denotes of
T12824 18670-18671 SYM denotes ∼
T12825 18671-18673 CD denotes 40
T12826 18673-18674 NN denotes %
T12827 18674-18676 , denotes ,
T12828 18676-18679 CC denotes and
T12829 18679-18681 , denotes ,
T12830 18681-18683 IN denotes as
T12831 18684-18692 VBN denotes expected
T12833 18693-18697 IN denotes from
T12834 18698-18700 NN denotes ES
T12835 18701-18705 NN denotes cell
T12836 18706-18717 NNS denotes experiments
T12837 18717-18719 , denotes ,
T12832 18719-18728 VBD denotes expressed
T12838 18729-18730 DT denotes a
T12840 18731-18739 NN denotes p53ΔPGFP
T12839 18740-18747 NN denotes protein
T12841 18748-18750 IN denotes at
T12842 18751-18755 RB denotes much
T12843 18756-18761 JJR denotes lower
T12844 18762-18768 NNS denotes levels
T12845 18769-18773 IN denotes than
T12846 18774-18779 NN denotes p53WT
T12847 18780-18781 -LRB- denotes (
T12849 18781-18787 NN denotes Figure
T12848 18788-18790 NN denotes 5B
T12850 18790-18791 -RRB- denotes )
T12851 18791-18792 . denotes .
T12852 18792-18874 sentence denotes Unlike WT MEFs, p53ΔP/ΔP MEFs are unable to arrest cycling after irradiation (5).
T12853 18793-18799 IN denotes Unlike
T12855 18800-18802 NN denotes WT
T12856 18803-18807 NNS denotes MEFs
T12857 18807-18809 , denotes ,
T12858 18809-18814 NN denotes p53ΔP
T12860 18814-18815 HYPH denotes /
T12859 18815-18817 NN denotes ΔP
T12861 18818-18822 NNS denotes MEFs
T12854 18823-18826 VBP denotes are
T12862 18827-18833 JJ denotes unable
T12863 18834-18836 TO denotes to
T12864 18837-18843 VB denotes arrest
T12865 18844-18851 VBG denotes cycling
T12866 18852-18857 IN denotes after
T12867 18858-18869 NN denotes irradiation
T12868 18870-18871 -LRB- denotes (
T12869 18871-18872 CD denotes 5
T12870 18872-18873 -RRB- denotes )
T12871 18873-18874 . denotes .
T12872 18874-19061 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 18875-18883 RB denotes Likewise
T12875 18883-18885 , denotes ,
T12876 18885-18887 PRP denotes we
T12874 18888-18893 VBD denotes found
T12877 18894-18898 IN denotes that
T12879 18899-18910 NN denotes irradiation
T12880 18911-18916 NNS denotes doses
T12881 18917-18921 WDT denotes that
T12882 18922-18930 VBD denotes arrested
T12883 18931-18938 NN denotes p53RMCE
T12885 18938-18939 HYPH denotes /
T12886 18939-18940 SYM denotes −
T12884 18941-18945 NNS denotes MEFs
T12887 18946-18947 -LRB- denotes (
T12888 18947-18952 WDT denotes which
T12889 18953-18960 VBP denotes express
T12890 18961-18962 DT denotes a
T12892 18963-18967 JJ denotes wild
T12894 18967-18968 HYPH denotes -
T12893 18968-18972 NN denotes type
T12891 18973-18976 NN denotes p53
T12895 18977-18981 IN denotes from
T12896 18982-18985 DT denotes the
T12898 18986-18990 NN denotes RMCE
T12900 18990-18991 HYPH denotes -
T12899 18991-18996 JJ denotes ready
T12897 18997-19002 NN denotes locus
T12901 19002-19004 , denotes ,
T12902 19004-19007 VBP denotes see
T12903 19008-19014 NN denotes Figure
T12904 19015-19016 CD denotes 2
T12905 19016-19017 -RRB- denotes )
T12906 19018-19021 VBD denotes did
T12907 19022-19025 RB denotes not
T12878 19026-19032 VB denotes arrest
T12908 19033-19041 NN denotes p53ΔPGFP
T12910 19041-19042 HYPH denotes /
T12911 19042-19043 SYM denotes −
T12909 19044-19048 NNS denotes MEFs
T12912 19049-19050 -LRB- denotes (
T12914 19050-19056 NN denotes Figure
T12913 19057-19059 NN denotes 5C
T12915 19059-19060 -RRB- denotes )
T12916 19060-19061 . denotes .
T12917 19061-19175 sentence denotes These data show that a single RMCE-ASAP reaction in heterozygous MEFs enables detection of a recessive phenotype.
T12918 19062-19067 DT denotes These
T12919 19068-19072 NNS denotes data
T12920 19073-19077 VBP denotes show
T12921 19078-19082 IN denotes that
T12923 19083-19084 DT denotes a
T12925 19085-19091 JJ denotes single
T12926 19092-19096 NN denotes RMCE
T12928 19096-19097 HYPH denotes -
T12927 19097-19101 NN denotes ASAP
T12924 19102-19110 NN denotes reaction
T12929 19111-19113 IN denotes in
T12930 19114-19126 JJ denotes heterozygous
T12931 19127-19131 NNS denotes MEFs
T12922 19132-19139 VBZ denotes enables
T12932 19140-19149 NN denotes detection
T12933 19150-19152 IN denotes of
T12934 19153-19154 DT denotes a
T12936 19155-19164 JJ denotes recessive
T12935 19165-19174 NN denotes phenotype
T12937 19174-19175 . denotes .
T12938 19175-19378 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 19176-19179 DT denotes The
T12940 19180-19187 NNS denotes results
T12941 19188-19195 VBP denotes confirm
T12942 19196-19200 IN denotes that
T12944 19201-19209 VBG denotes deleting
T12945 19210-19213 DT denotes the
T12947 19214-19221 NN denotes proline
T12948 19222-19226 JJ denotes rich
T12946 19227-19233 NN denotes domain
T12943 19234-19239 VBZ denotes leads
T12949 19240-19242 IN denotes to
T12950 19243-19247 RBR denotes less
T12951 19248-19254 JJ denotes active
T12952 19255-19258 NN denotes p53
T12953 19259-19263 IN denotes with
T12954 19264-19272 JJ denotes impaired
T12956 19273-19277 NN denotes cell
T12957 19278-19283 NN denotes cycle
T12955 19284-19291 NN denotes control
T12958 19291-19293 , denotes ,
T12959 19293-19296 CC denotes and
T12960 19297-19301 RB denotes also
T12961 19302-19310 VBP denotes indicate
T12962 19311-19315 IN denotes that
T12964 19316-19317 DT denotes a
T12966 19318-19321 NN denotes GFP
T12967 19322-19323 NN denotes C
T12968 19323-19324 HYPH denotes -
T12969 19324-19332 JJ denotes terminal
T12965 19333-19339 NN denotes fusion
T12970 19340-19343 MD denotes can
T12971 19344-19356 RB denotes dramatically
T12963 19357-19362 VB denotes alter
T12972 19363-19366 NN denotes p53
T12973 19367-19377 NN denotes regulation
T12974 19377-19378 . denotes .
T12975 19378-19428 sentence denotes A summary of our results is presented in Table 1.
T12976 19379-19380 DT denotes A
T12977 19381-19388 NN denotes summary
T12979 19389-19391 IN denotes of
T12980 19392-19395 PRP$ denotes our
T12981 19396-19403 NNS denotes results
T12982 19404-19406 VBZ denotes is
T12978 19407-19416 VBN denotes presented
T12983 19417-19419 IN denotes in
T12984 19420-19425 NN denotes Table
T12985 19426-19427 CD denotes 1
T12986 19427-19428 . denotes .
T12987 19428-19642 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 19429-19434 DT denotes These
T12989 19435-19439 NNS denotes data
T12990 19440-19446 VBP denotes report
T12991 19447-19450 DT denotes the
T12992 19451-19462 NN denotes development
T12993 19463-19466 CC denotes and
T12994 19467-19481 NN denotes implementation
T12995 19482-19484 IN denotes of
T12996 19485-19487 DT denotes an
T12998 19488-19496 VBN denotes improved
T12999 19497-19501 NN denotes RMCE
T12997 19502-19510 NN denotes approach
T13000 19511-19515 WDT denotes that
T13001 19516-19523 VBZ denotes enables
T13002 19524-19533 JJ denotes efficient
T13004 19534-19540 NN denotes allele
T13003 19541-19553 NN denotes modification
T13005 19554-19556 IN denotes in
T13006 19557-19559 NN denotes ES
T13007 19560-19565 NNS denotes cells
T13008 19566-19568 TO denotes to
T13009 19569-19577 VB denotes generate
T13010 19578-19582 NNS denotes mice
T13011 19583-19586 CC denotes and
T13012 19587-19589 IN denotes in
T13014 19590-19602 JJ denotes heterozygous
T13015 19603-19607 NNS denotes MEFs
T13016 19608-19610 TO denotes to
T13013 19611-19621 VB denotes accelerate
T13017 19622-19632 JJ denotes phenotypic
T13018 19633-19641 NNS denotes analyses
T13019 19641-19642 . denotes .
T13020 19642-19905 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 19643-19646 DT denotes The
T13022 19647-19654 NN denotes success
T13024 19655-19657 IN denotes of
T13025 19658-19662 NN denotes RMCE
T13027 19662-19663 HYPH denotes -
T13026 19663-19667 NN denotes ASAP
T13023 19668-19674 VBN denotes relied
T13028 19675-19677 IN denotes on
T13029 19678-19681 DT denotes the
T13031 19682-19692 VBN denotes integrated
T13030 19693-19696 NN denotes use
T13032 19697-19699 IN denotes of
T13033 19700-19708 VBN denotes inverted
T13035 19709-19721 JJ denotes heterologous
T13036 19722-19726 NN denotes loxP
T13034 19727-19732 NNS denotes sites
T13037 19732-19734 , denotes ,
T13038 19734-19735 DT denotes a
T13040 19736-19744 JJ denotes positive
T13042 19744-19745 HYPH denotes /
T13041 19745-19753 JJ denotes negative
T13043 19754-19763 NN denotes selection
T13039 19764-19770 NN denotes marker
T13044 19771-19775 WDT denotes that
T13045 19776-19785 VBZ denotes preserves
T13046 19786-19789 DT denotes the
T13048 19790-19798 NN denotes germline
T13047 19799-19807 NN denotes capacity
T13049 19808-19810 IN denotes of
T13050 19811-19813 NN denotes ES
T13051 19814-19819 NNS denotes cells
T13052 19819-19821 , denotes ,
T13053 19821-19824 CC denotes and
T13054 19824-19826 , denotes ,
T13055 19826-19829 IN denotes for
T13057 19830-19837 JJ denotes somatic
T13058 19838-19843 NNS denotes cells
T13059 19843-19845 , denotes ,
T13060 19845-19848 DT denotes the
T13056 19849-19858 NN denotes existence
T13061 19859-19861 IN denotes of
T13062 19862-19863 DT denotes a
T13064 19864-19869 VB denotes knock
T13065 19869-19870 HYPH denotes -
T13066 19870-19873 RP denotes out
T13063 19874-19880 NN denotes allele
T13067 19881-19883 IN denotes of
T13068 19884-19887 DT denotes the
T13069 19888-19892 NN denotes gene
T13070 19893-19895 IN denotes of
T13071 19896-19904 NN denotes interest
T13072 19904-19905 . denotes .
T13073 19905-20092 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 19906-19911 DT denotes These
T13075 19912-19927 NNS denotes characteristics
T13077 19928-19934 MD denotes should
T13076 19935-19939 VB denotes make
T13078 19940-19944 NN denotes RMCE
T13080 19944-19945 HYPH denotes -
T13079 19945-19949 NN denotes ASAP
T13082 19950-19951 DT denotes a
T13083 19952-19958 JJ denotes robust
T13084 19959-19962 CC denotes and
T13085 19963-19970 JJ denotes general
T13081 19971-19981 NN denotes technology
T13086 19982-19985 IN denotes for
T13087 19986-19994 NN denotes analysis
T13088 19995-19997 IN denotes of
T13089 19998-20007 JJ denotes mammalian
T13090 20008-20013 NNS denotes genes
T13091 20014-20019 IN denotes under
T13092 20020-20030 NNS denotes conditions
T13093 20031-20035 WDT denotes that
T13094 20036-20044 VBP denotes preserve
T13095 20045-20051 JJ denotes normal
T13097 20052-20059 NN denotes control
T13096 20060-20070 NNS denotes mechanisms
T13098 20071-20073 IN denotes in
T13099 20074-20083 JJ denotes different
T13100 20084-20091 NNS denotes tissues
T13101 20091-20092 . denotes .
T13102 20092-20252 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 20093-20095 IN denotes In
T13105 20096-20104 NN denotes addition
T13106 20104-20106 , denotes ,
T13107 20106-20110 NN denotes RMCE
T13109 20110-20111 HYPH denotes -
T13108 20111-20115 NN denotes ASAP
T13110 20116-20121 MD denotes could
T13111 20122-20124 VB denotes be
T13104 20125-20129 VBN denotes used
T13112 20130-20132 TO denotes to
T13113 20133-20141 VB denotes generate
T13114 20142-20154 JJ denotes fibroblastic
T13116 20155-20159 NN denotes cell
T13115 20160-20165 NNS denotes lines
T13117 20166-20174 VBN denotes tailored
T13118 20175-20178 IN denotes for
T13119 20179-20182 DT denotes the
T13121 20183-20191 VBN denotes repeated
T13120 20192-20201 NN denotes targeting
T13122 20202-20204 IN denotes of
T13123 20205-20211 RB denotes widely
T13124 20212-20219 VBN denotes studied
T13125 20220-20225 NNS denotes genes
T13126 20226-20227 -LRB- denotes (
T13127 20227-20230 NN denotes p53
T13128 20230-20232 , denotes ,
T13129 20232-20233 NN denotes c
T13131 20233-20234 HYPH denotes -
T13130 20234-20237 NN denotes myc
T13132 20237-20239 , denotes ,
T13133 20239-20241 NN denotes NF
T13135 20241-20242 HYPH denotes -
T13134 20242-20244 NN denotes KB
T13136 20244-20246 , denotes ,
T13137 20246-20250 FW denotes etc.
T13138 20250-20251 -RRB- denotes )
T13139 20251-20252 . denotes .
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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+
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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.
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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