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PMC:1540739 / 6128-14129 JSONTXT 5 Projects

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Id Subject Object Predicate Lexical cue
T4611 23-32 NN denotes Targeting
T4612 33-42 NN denotes construct
T4613 43-46 IN denotes for
T4614 47-48 DT denotes a
T4616 49-52 NN denotes p53
T4617 53-57 NN denotes RMCE
T4619 57-58 HYPH denotes -
T4618 58-63 JJ denotes ready
T4615 64-69 NN denotes locus
T4620 69-183 sentence denotes Details for plasmid construction are available upon request owing to space limitations mandating a brief outline.
T4621 70-77 NNS denotes Details
T4623 78-81 IN denotes for
T4624 82-89 NN denotes plasmid
T4625 90-102 NN denotes construction
T4622 103-106 VBP denotes are
T4626 107-116 JJ denotes available
T4627 117-121 IN denotes upon
T4628 122-129 NN denotes request
T4629 130-135 VBG denotes owing
T4630 136-138 IN denotes to
T4631 139-144 NN denotes space
T4632 145-156 NNS denotes limitations
T4633 157-166 VBG denotes mandating
T4634 167-168 DT denotes a
T4636 169-174 JJ denotes brief
T4635 175-182 NN denotes outline
T4637 182-183 . denotes .
T4638 183-329 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 184-186 PRP denotes We
T4640 187-194 VBD denotes started
T4641 195-199 IN denotes from
T4642 200-201 DT denotes a
T4644 202-209 NN denotes plasmid
T4645 210-212 NN denotes L3
T4646 212-213 HYPH denotes -
T4643 213-215 NN denotes 1L
T4647 216-226 VBG denotes containing
T4648 227-239 JJ denotes heterologous
T4650 240-244 NN denotes loxP
T4649 245-250 NNS denotes sites
T4651 251-252 -LRB- denotes (
T4653 252-254 NN denotes L3
T4654 255-257 VBZ denotes is
T4655 258-261 DT denotes the
T4657 262-268 JJ denotes mutant
T4656 269-276 NN denotes loxP257
T4658 277-285 RB denotes recently
T4659 286-295 VBN denotes described
T4660 296-297 -LRB- denotes (
T4661 297-299 CD denotes 14
T4662 299-300 -RRB- denotes )
T4663 300-302 , denotes ,
T4664 302-304 NN denotes 1L
T4652 305-307 VBZ denotes is
T4665 308-310 DT denotes an
T4667 311-319 VBN denotes inverted
T4668 320-322 NN denotes WT
T4666 323-327 NN denotes loxP
T4669 327-328 -RRB- denotes )
T4670 328-329 . denotes .
T4671 329-469 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 330-333 DT denotes The
T4674 334-336 NN denotes WT
T4673 337-341 NN denotes loxP
T4676 342-345 CC denotes and
T4677 346-353 NN denotes loxP257
T4675 354-360 VBP denotes differ
T4679 361-363 IN denotes in
T4680 364-369 PRP$ denotes their
T4682 370-376 NN denotes spacer
T4681 377-386 NNS denotes sequences
T4683 386-388 : denotes :
T4684 388-391 DT denotes the
T4686 392-398 NN denotes spacer
T4685 399-407 NN denotes sequence
T4678 408-410 VBZ denotes is
T4687 411-412 CD denotes 5
T4688 412-413 SYM denotes ′
T4689 413-414 HYPH denotes -
T4690 414-422 JJ denotes ATGTATGC
T4691 422-423 HYPH denotes -
T4692 423-424 CD denotes 3
T4693 424-425 SYM denotes ′
T4694 426-429 IN denotes for
T4695 430-432 NN denotes WT
T4696 433-437 NN denotes loxP
T4697 438-441 CC denotes and
T4698 442-443 CD denotes 5
T4700 443-444 SYM denotes ′
T4701 444-445 HYPH denotes -
T4699 445-453 NN denotes AAGTCTCC
T4702 453-454 HYPH denotes -
T4703 454-455 CD denotes 3
T4704 455-456 SYM denotes ′
T4705 457-460 IN denotes for
T4706 461-468 NN denotes loxP257
T4707 468-469 . denotes .
T4708 469-773 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 470-473 DT denotes The
T4711 474-479 CD denotes three
T4710 480-489 NNS denotes mutations
T4713 490-492 IN denotes in
T4714 493-496 DT denotes the
T4716 497-504 NN denotes loxP257
T4717 505-511 NN denotes spacer
T4715 512-520 NN denotes sequence
T4712 521-528 VBP denotes prevent
T4719 529-531 PRP denotes it
T4721 532-534 TO denotes to
T4720 535-544 VB denotes recombine
T4722 545-549 IN denotes with
T4723 550-552 NN denotes WT
T4724 553-557 NN denotes loxP
T4725 557-559 , denotes ,
T4726 559-567 VBG denotes ensuring
T4727 568-577 JJ denotes efficient
T4728 578-582 NN denotes RMCE
T4729 583-585 IN denotes in
T4730 586-593 JJ denotes several
T4732 594-598 NN denotes cell
T4731 599-604 NNS denotes lines
T4733 604-606 : denotes :
T4734 606-614 JJ denotes accurate
T4735 615-619 NN denotes RMCE
T4736 620-624 IN denotes with
T4737 625-630 DT denotes these
T4739 631-635 NN denotes loxP
T4738 636-641 NNS denotes sites
T4718 642-650 VBD denotes occurred
T4740 651-655 IN denotes with
T4741 656-658 DT denotes an
T4743 659-666 JJ denotes average
T4742 667-676 NN denotes frequency
T4744 677-679 IN denotes of
T4745 680-682 CD denotes 81
T4746 682-683 NN denotes %
T4747 684-686 IN denotes at
T4748 687-690 CD denotes two
T4749 691-695 NNS denotes loci
T4750 696-698 IN denotes in
T4751 699-702 NN denotes CHO
T4752 703-708 NNS denotes cells
T4753 709-712 CC denotes and
T4754 713-715 DT denotes an
T4756 716-723 JJ denotes average
T4755 724-733 NN denotes frequency
T4757 734-736 IN denotes of
T4758 737-739 CD denotes 69
T4759 739-740 NN denotes %
T4760 741-743 IN denotes at
T4761 744-748 CD denotes four
T4762 749-753 NNS denotes loci
T4763 754-756 IN denotes in
T4764 757-761 NN denotes Hela
T4765 762-767 NNS denotes cells
T4766 768-769 -LRB- denotes (
T4767 769-771 CD denotes 14
T4768 771-772 -RRB- denotes )
T4769 772-773 . denotes .
T4770 773-897 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 774-777 DT denotes The
T4773 778-780 NN denotes L3
T4775 780-781 HYPH denotes -
T4774 781-783 NN denotes 1L
T4772 784-791 NN denotes plasmid
T4777 792-795 VBD denotes was
T4778 796-801 RB denotes first
T4776 802-810 VBN denotes modified
T4779 811-813 TO denotes to
T4780 814-821 VB denotes include
T4781 822-823 DT denotes a
T4782 824-828 NN denotes ClaI
T4783 829-832 CC denotes and
T4784 833-834 DT denotes a
T4786 835-839 NN denotes FseI
T4785 840-844 NN denotes site
T4787 845-852 IN denotes between
T4788 853-856 DT denotes the
T4790 857-861 NN denotes LoxP
T4789 862-867 NNS denotes sites
T4791 867-869 , denotes ,
T4792 869-876 VBG denotes leading
T4793 877-879 IN denotes to
T4794 880-887 NN denotes plasmid
T4796 888-890 NN denotes L3
T4797 890-891 HYPH denotes -
T4798 891-893 NN denotes CF
T4799 893-894 HYPH denotes -
T4795 894-896 NN denotes 1L
T4800 896-897 . denotes .
T4801 897-1159 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 898-900 PRP denotes We
T4804 901-905 RB denotes next
T4803 906-914 VBD denotes modified
T4805 915-916 DT denotes a
T4807 917-924 NN denotes puroΔTK
T4806 925-932 NN denotes plasmid
T4808 933-934 -LRB- denotes (
T4809 934-939 NN denotes YTC37
T4810 939-941 , denotes ,
T4811 941-942 DT denotes a
T4813 943-947 NN denotes kind
T4812 948-952 NN denotes gift
T4814 953-957 IN denotes from
T4815 958-960 NNP denotes A.
T4816 961-968 NNP denotes Bradley
T4817 968-969 -RRB- denotes )
T4818 970-972 IN denotes by
T4819 973-978 VBG denotes using
T4820 979-995 NNS denotes oligonucleotides
T4821 996-998 TO denotes to
T4822 999-1006 VB denotes destroy
T4823 1007-1008 DT denotes a
T4825 1009-1013 NN denotes NotI
T4824 1014-1018 NN denotes site
T4826 1019-1029 RB denotes downstream
T4827 1030-1032 IN denotes of
T4828 1033-1036 DT denotes the
T4830 1037-1044 NN denotes puroΔTK
T4829 1045-1049 NN denotes gene
T4831 1050-1053 CC denotes and
T4832 1054-1063 VB denotes introduce
T4833 1064-1065 DT denotes a
T4835 1066-1070 NN denotes NotI
T4834 1071-1075 NN denotes site
T4836 1076-1084 RB denotes upstream
T4837 1084-1086 , denotes ,
T4838 1086-1089 CC denotes and
T4839 1090-1091 DT denotes a
T4841 1092-1096 NN denotes FseI
T4840 1097-1101 NN denotes site
T4842 1102-1112 RB denotes downstream
T4843 1113-1115 IN denotes of
T4844 1116-1119 DT denotes the
T4845 1120-1124 NN denotes gene
T4846 1125-1126 -LRB- denotes (
T4847 1126-1133 VBG denotes leading
T4848 1134-1136 IN denotes to
T4849 1137-1144 NN denotes plasmid
T4851 1145-1147 NN denotes CN
T4852 1147-1148 HYPH denotes -
T4853 1148-1155 NN denotes PuroΔTK
T4854 1155-1156 HYPH denotes -
T4850 1156-1157 NN denotes F
T4855 1157-1158 -RRB- denotes )
T4856 1158-1159 . denotes .
T4857 1159-1407 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 1160-1164 RB denotes Next
T4860 1164-1166 , denotes ,
T4861 1166-1167 DT denotes a
T4863 1168-1172 NN denotes PmlI
T4865 1172-1173 HYPH denotes -
T4864 1173-1177 NN denotes MfeI
T4866 1178-1181 CD denotes 6.3
T4867 1182-1184 NN denotes kb
T4862 1185-1193 NN denotes fragment
T4868 1194-1198 IN denotes from
T4869 1199-1204 NN denotes Trp53
T4870 1205-1208 VBD denotes was
T4859 1209-1218 VBN denotes subcloned
T4871 1219-1221 IN denotes in
T4872 1222-1223 DT denotes a
T4874 1224-1232 VBN denotes modified
T4875 1233-1244 NN denotes pBluescript
T4873 1245-1250 NN denotes KSII+
T4876 1251-1252 -LRB- denotes (
T4877 1252-1255 NN denotes pBS
T4878 1255-1257 , denotes ,
T4879 1257-1267 NNP denotes Stratagene
T4880 1267-1268 -RRB- denotes )
T4881 1268-1270 , denotes ,
T4882 1270-1273 CC denotes and
T4883 1274-1277 DT denotes the
T4885 1278-1287 VBG denotes resulting
T4884 1288-1295 NN denotes plasmid
T4887 1296-1299 VBD denotes was
T4886 1300-1308 VBN denotes digested
T4888 1309-1313 IN denotes with
T4889 1314-1318 NN denotes SwaI
T4890 1319-1321 TO denotes to
T4891 1322-1331 VB denotes introduce
T4892 1332-1334 DT denotes an
T4894 1335-1339 NN denotes EagI
T4893 1340-1344 NN denotes site
T4895 1344-1346 , denotes ,
T4896 1346-1353 VBG denotes leading
T4897 1354-1356 IN denotes to
T4898 1357-1366 NN denotes p53PmlEag
T4899 1366-1368 , denotes ,
T4900 1368-1369 DT denotes a
T4901 1370-1377 NN denotes plasmid
T4902 1378-1388 VBG denotes containing
T4903 1389-1394 NNS denotes exons
T4905 1395-1396 CD denotes 2
T4906 1396-1397 HYPH denotes –
T4904 1397-1399 CD denotes 11
T4907 1400-1402 IN denotes of
T4908 1403-1406 NN denotes p53
T4909 1406-1407 . denotes .
T4910 1407-1652 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 1408-1410 PRP denotes We
T4913 1411-1415 RB denotes then
T4912 1416-1424 VBD denotes inserted
T4914 1425-1426 DT denotes a
T4916 1427-1430 CD denotes 5.5
T4917 1431-1433 NN denotes kb
T4918 1434-1438 NN denotes ClaI
T4920 1438-1439 HYPH denotes -
T4919 1439-1443 NN denotes EagI
T4915 1444-1452 NN denotes fragment
T4921 1453-1457 IN denotes from
T4922 1458-1467 NN denotes p53PmlEag
T4923 1468-1470 IN denotes in
T4924 1471-1478 NN denotes plasmid
T4926 1479-1481 NN denotes CN
T4927 1481-1482 HYPH denotes -
T4928 1482-1489 NN denotes PuroΔTK
T4929 1489-1490 HYPH denotes -
T4925 1490-1491 NN denotes F
T4930 1492-1500 VBN denotes digested
T4931 1501-1503 IN denotes by
T4932 1504-1508 NN denotes ClaI
T4933 1509-1512 CC denotes and
T4934 1513-1517 NN denotes NotI
T4935 1517-1519 , denotes ,
T4936 1519-1522 CC denotes and
T4937 1523-1531 VBD denotes inserted
T4938 1532-1535 DT denotes the
T4940 1536-1545 VBG denotes resulting
T4939 1546-1554 NN denotes fragment
T4941 1555-1562 IN denotes between
T4942 1563-1566 DT denotes the
T4944 1567-1571 NN denotes loxP
T4943 1572-1577 NNS denotes sites
T4945 1578-1580 IN denotes of
T4946 1581-1583 NN denotes L3
T4948 1583-1584 HYPH denotes -
T4949 1584-1586 NN denotes CF
T4950 1586-1587 HYPH denotes -
T4947 1587-1589 NN denotes 1L
T4951 1590-1592 IN denotes by
T4952 1593-1597 NN denotes ClaI
T4954 1598-1601 CC denotes and
T4955 1602-1606 NN denotes FseI
T4953 1607-1616 NN denotes digestion
T4956 1616-1618 , denotes ,
T4957 1618-1625 VBG denotes leading
T4958 1626-1628 IN denotes to
T4959 1629-1631 NN denotes L3
T4961 1631-1632 HYPH denotes -
T4962 1632-1648 NN denotes p53PmlEagPuroΔTK
T4963 1648-1649 HYPH denotes -
T4960 1649-1651 NN denotes 1L
T4964 1651-1652 . denotes .
T4965 1652-2242 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 1653-1655 PRP denotes We
T4968 1656-1660 RB denotes next
T4967 1661-1671 VBD denotes engineered
T4970 1672-1673 DT denotes a
T4971 1674-1681 NN denotes plasmid
T4972 1682-1692 VBG denotes containing
T4973 1693-1696 DT denotes the
T4974 1697-1703 NN denotes region
T4975 1704-1707 IN denotes for
T4976 1708-1709 CD denotes 3
T4977 1709-1710 SYM denotes ′
T4978 1710-1711 SYM denotes -
T4979 1711-1719 JJ denotes homology
T4980 1720-1730 RB denotes downstream
T4981 1731-1733 IN denotes of
T4982 1734-1737 DT denotes the
T4984 1738-1741 NN denotes p53
T4983 1742-1746 NN denotes gene
T4985 1747-1750 CC denotes and
T4986 1751-1754 DT denotes the
T4988 1755-1758 NN denotes DTA
T4987 1759-1763 NN denotes gene
T4989 1764-1766 IN denotes in
T4990 1767-1770 CD denotes two
T4991 1771-1776 NNS denotes steps
T4992 1776-1778 : denotes :
T4993 1778-1779 -LRB- denotes (
T4994 1779-1780 LS denotes i
T4995 1780-1781 -RRB- denotes )
T4996 1782-1784 PRP denotes we
T4969 1785-1794 VBD denotes performed
T4997 1795-1796 DT denotes a
T4999 1797-1802 CD denotes three
T5001 1802-1803 HYPH denotes -
T5000 1803-1806 NN denotes way
T4998 1807-1815 NN denotes ligation
T5002 1816-1823 IN denotes between
T5003 1824-1825 DT denotes a
T5005 1826-1834 VBN denotes modified
T5004 1835-1838 NN denotes pBS
T5006 1839-1847 VBN denotes digested
T5007 1848-1850 IN denotes by
T5008 1851-1858 NN denotes HindIII
T5009 1859-1862 CC denotes and
T5010 1863-1867 NN denotes NotI
T5011 1867-1869 , denotes ,
T5012 1869-1870 DT denotes a
T5014 1871-1878 NN denotes HindIII
T5016 1878-1879 HYPH denotes -
T5015 1879-1884 NN denotes EcoRI
T5013 1885-1893 NN denotes fragment
T5017 1894-1898 IN denotes from
T5018 1899-1904 NN denotes Trp53
T5019 1905-1908 IN denotes for
T5020 1909-1910 CD denotes 3
T5022 1910-1911 SYM denotes ′
T5021 1911-1919 NN denotes homology
T5023 1920-1923 CC denotes and
T5024 1924-1926 DT denotes an
T5026 1927-1932 NN denotes EcoRI
T5028 1932-1933 HYPH denotes -
T5027 1933-1937 NN denotes NotI
T5025 1938-1946 NN denotes fragment
T5029 1947-1957 VBG denotes containing
T5030 1958-1961 DT denotes the
T5032 1962-1965 NN denotes DTA
T5031 1966-1970 NN denotes gene
T5033 1970-1972 , denotes ,
T5034 1972-1976 IN denotes from
T5035 1977-1984 NN denotes plasmid
T5036 1985-1994 NN denotes pgkdtabpa
T5037 1995-1996 -LRB- denotes (
T5038 1996-2000 JJ denotes kind
T5039 2001-2005 NN denotes gift
T5040 2006-2008 IN denotes of
T5041 2009-2011 NNP denotes P.
T5042 2012-2019 NNP denotes Soriano
T5043 2019-2020 -RRB- denotes )
T5044 2020-2022 , denotes ,
T5045 2022-2029 VBG denotes leading
T5046 2030-2032 IN denotes to
T5047 2033-2040 NN denotes plasmid
T5049 2041-2042 CD denotes 3
T5050 2042-2043 SYM denotes ′
T5051 2044-2045 SYM denotes +
T5048 2046-2049 NN denotes DTA
T5052 2050-2053 CC denotes and
T5053 2054-2055 -LRB- denotes (
T5054 2055-2057 LS denotes ii
T5056 2057-2058 -RRB- denotes )
T5057 2059-2066 IN denotes because
T5059 2067-2070 DT denotes the
T5061 2071-2077 NN denotes Bsu36I
T5063 2077-2078 HYPH denotes -
T5062 2078-2083 NN denotes EcoRI
T5060 2084-2090 NN denotes region
T5064 2091-2101 RB denotes downstream
T5065 2102-2104 IN denotes of
T5066 2105-2108 NN denotes p53
T5058 2109-2117 VBZ denotes contains
T5067 2118-2128 JJ denotes repetitive
T5068 2129-2138 NNS denotes sequences
T5069 2139-2140 -LRB- denotes (
T5070 2140-2142 NNP denotes F.
T5071 2143-2149 NNP denotes Toledo
T5072 2150-2153 CC denotes and
T5073 2154-2156 NNP denotes G.
T5074 2157-2159 NNP denotes M.
T5075 2160-2164 NNP denotes Wahl
T5076 2164-2166 , denotes ,
T5077 2166-2177 JJ denotes unpublished
T5078 2178-2182 NNS denotes data
T5079 2182-2183 -RRB- denotes )
T5080 2183-2185 , denotes ,
T5081 2185-2187 PRP denotes we
T5082 2188-2193 RB denotes later
T5055 2194-2201 VBD denotes deleted
T5083 2202-2206 DT denotes this
T5084 2207-2213 NN denotes region
T5085 2213-2215 , denotes ,
T5086 2215-2217 TO denotes to
T5087 2218-2224 VB denotes obtain
T5088 2225-2232 NN denotes plasmid
T5090 2233-2234 CD denotes 3
T5091 2234-2235 SYM denotes ′
T5092 2236-2237 SYM denotes +
T5089 2238-2241 NN denotes DTA
T5093 2241-2242 . denotes .
T5094 2242-2365 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 2243-2246 DT denotes The
T5097 2247-2248 CD denotes 5
T5098 2248-2249 SYM denotes ′
T5096 2250-2258 NN denotes homology
T5099 2259-2267 VBZ denotes consists
T5100 2268-2270 IN denotes of
T5101 2271-2272 DT denotes a
T5103 2273-2276 CD denotes 3.4
T5104 2277-2279 NN denotes kb
T5106 2279-2280 HYPH denotes -
T5105 2280-2284 JJ denotes long
T5107 2285-2290 NN denotes BamHI
T5109 2290-2291 HYPH denotes -
T5108 2291-2295 NN denotes PmlI
T5102 2296-2304 NN denotes fragment
T5110 2305-2309 IN denotes from
T5111 2310-2316 NN denotes intron
T5112 2317-2318 CD denotes 1
T5113 2319-2321 IN denotes of
T5114 2322-2325 NN denotes p53
T5115 2326-2332 VBN denotes cloned
T5116 2333-2335 IN denotes in
T5117 2336-2337 DT denotes a
T5119 2338-2346 VBN denotes modified
T5118 2347-2350 NN denotes pBS
T5120 2351-2352 -LRB- denotes (
T5121 2352-2359 NN denotes plasmid
T5122 2360-2362 NN denotes p5
T5123 2362-2363 SYM denotes ′
T5124 2363-2364 -RRB- denotes )
T5125 2364-2365 . denotes .
T5126 2365-2506 sentence denotes Finally, appropriate fragments from plasmids p5′, L3-p53PmlEagPuroΔTK-1L, and 3′ + DTA were assembled in a modified pSP72 plasmid (Promega).
T5127 2366-2373 RB denotes Finally
T5129 2373-2375 , denotes ,
T5130 2375-2386 JJ denotes appropriate
T5131 2387-2396 NNS denotes fragments
T5132 2397-2401 IN denotes from
T5133 2402-2410 NNS denotes plasmids
T5134 2411-2413 NN denotes p5
T5135 2413-2414 SYM denotes ′
T5136 2414-2416 , denotes ,
T5137 2416-2418 NN denotes L3
T5139 2418-2419 HYPH denotes -
T5140 2419-2435 NN denotes p53PmlEagPuroΔTK
T5141 2435-2436 HYPH denotes -
T5138 2436-2438 NN denotes 1L
T5142 2438-2440 , denotes ,
T5143 2440-2443 CC denotes and
T5144 2444-2445 CD denotes 3
T5146 2445-2446 SYM denotes ′
T5147 2447-2448 SYM denotes +
T5145 2449-2452 NN denotes DTA
T5148 2453-2457 VBD denotes were
T5128 2458-2467 VBN denotes assembled
T5149 2468-2470 IN denotes in
T5150 2471-2472 DT denotes a
T5152 2473-2481 VBN denotes modified
T5153 2482-2487 NN denotes pSP72
T5151 2488-2495 NN denotes plasmid
T5154 2496-2497 -LRB- denotes (
T5155 2497-2504 NNP denotes Promega
T5156 2504-2505 -RRB- denotes )
T5157 2505-2506 . denotes .
T5158 2506-2752 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 2507-2514 NN denotes Plasmid
T5160 2515-2519 NN denotes Flox
T5162 2519-2521 , denotes ,
T5163 2521-2524 DT denotes the
T5165 2525-2534 VBG denotes resulting
T5166 2535-2544 NN denotes targeting
T5164 2545-2554 NN denotes construct
T5167 2554-2556 , denotes ,
T5168 2556-2559 VBD denotes was
T5161 2560-2568 VBN denotes verified
T5169 2569-2571 IN denotes by
T5170 2572-2583 NN denotes restriction
T5171 2584-2592 NN denotes analysis
T5172 2592-2594 , denotes ,
T5173 2594-2598 RB denotes then
T5174 2599-2608 VBN denotes sequenced
T5175 2609-2614 VBG denotes using
T5176 2615-2617 CD denotes 30
T5177 2618-2625 NNS denotes primers
T5178 2626-2632 VBN denotes chosen
T5179 2633-2635 TO denotes to
T5181 2636-2645 RB denotes precisely
T5180 2646-2652 VB denotes verify
T5182 2653-2656 DT denotes all
T5184 2657-2660 NN denotes p53
T5185 2661-2667 NN denotes coding
T5183 2668-2677 NNS denotes sequences
T5186 2677-2679 , denotes ,
T5187 2679-2682 DT denotes all
T5189 2683-2687 NN denotes exon
T5191 2687-2688 HYPH denotes –
T5190 2688-2694 NN denotes intron
T5188 2695-2704 NNS denotes junctions
T5192 2705-2708 CC denotes and
T5193 2709-2712 DT denotes the
T5194 2713-2722 NNS denotes sequences
T5195 2723-2725 IN denotes at
T5196 2726-2729 CC denotes and
T5197 2730-2736 IN denotes around
T5198 2737-2740 DT denotes the
T5200 2741-2745 NN denotes loxP
T5199 2746-2751 NNS denotes sites
T5201 2751-2752 . denotes .
T5752 2754-2762 NN denotes Exchange
T5753 2763-2773 NNS denotes constructs
T5754 2773-2775 : denotes :
T5755 2775-2781 VBG denotes making
T5756 2782-2785 DT denotes the
T5757 2786-2792 NN denotes p53GFP
T5759 2793-2796 CC denotes and
T5760 2797-2800 NN denotes p53
T5762 2800-2801 NN denotes Δ
T5761 2801-2805 NN denotes PGFP
T5758 2806-2814 NNS denotes plasmids
T5763 2814-3038 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 2815-2817 TO denotes To
T5765 2818-2822 VB denotes make
T5767 2823-2824 DT denotes a
T5769 2825-2828 NN denotes p53
T5771 2828-2829 HYPH denotes -
T5770 2829-2832 NN denotes GFP
T5772 2833-2839 NN denotes fusion
T5768 2840-2847 NN denotes protein
T5773 2847-2849 , denotes ,
T5774 2849-2851 PRP denotes we
T5775 2852-2857 RB denotes first
T5766 2858-2867 VBD denotes subcloned
T5776 2868-2869 DT denotes a
T5778 2870-2875 NN denotes SacII
T5780 2875-2876 HYPH denotes -
T5779 2876-2883 NN denotes HindIII
T5777 2884-2892 NN denotes fragment
T5781 2893-2895 IN denotes of
T5782 2896-2899 DT denotes the
T5784 2900-2903 NN denotes p53
T5783 2904-2909 NN denotes locus
T5785 2910-2911 -LRB- denotes (
T5786 2911-2924 VBG denotes corresponding
T5787 2925-2927 IN denotes to
T5788 2928-2932 NN denotes part
T5789 2933-2935 IN denotes of
T5790 2936-2940 NN denotes exon
T5791 2941-2943 CD denotes 10
T5792 2944-2946 IN denotes to
T5793 2947-2956 NNS denotes sequences
T5794 2957-2967 RB denotes downstream
T5795 2968-2970 IN denotes of
T5796 2971-2974 DT denotes the
T5797 2975-2979 NN denotes gene
T5798 2979-2980 -RRB- denotes )
T5799 2981-2985 IN denotes into
T5800 2986-2989 NN denotes pBS
T5801 2989-2991 , denotes ,
T5802 2991-2995 RB denotes then
T5803 2996-3003 VBD denotes mutated
T5804 3004-3007 DT denotes the
T5806 3008-3015 NN denotes HindIII
T5805 3016-3020 NN denotes site
T5807 3021-3025 IN denotes into
T5808 3026-3027 DT denotes a
T5810 3028-3032 NN denotes FseI
T5809 3033-3037 NN denotes site
T5811 3037-3038 . denotes .
T5812 3038-3429 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 3039-3041 PRP denotes We
T5815 3042-3046 RB denotes next
T5814 3047-3054 VBD denotes mutated
T5816 3055-3058 DT denotes the
T5818 3059-3060 NN denotes C
T5820 3060-3061 HYPH denotes -
T5819 3061-3069 JJ denotes terminal
T5817 3070-3074 NN denotes part
T5821 3075-3077 IN denotes of
T5822 3078-3081 DT denotes the
T5824 3082-3085 NN denotes p53
T5823 3086-3090 NN denotes gene
T5825 3091-3093 IN denotes in
T5826 3094-3097 CD denotes two
T5827 3098-3104 NNS denotes rounds
T5828 3105-3107 IN denotes of
T5829 3108-3111 NN denotes PCR
T5830 3112-3123 NN denotes mutagenesis
T5831 3123-3125 , denotes ,
T5832 3125-3130 RB denotes first
T5833 3131-3135 IN denotes with
T5834 3136-3143 NNS denotes primers
T5836 3144-3145 CD denotes 5
T5837 3145-3146 SYM denotes ′
T5838 3146-3147 HYPH denotes -
T5835 3147-3183 NN denotes GGGCCTGACTCAGACGGATCCCCTCTGCATCCCGTC
T5839 3183-3184 HYPH denotes -
T5840 3184-3185 CD denotes 3
T5841 3185-3186 SYM denotes ′
T5842 3187-3190 CC denotes and
T5843 3191-3192 CD denotes 5
T5845 3192-3193 SYM denotes ′
T5846 3193-3194 HYPH denotes -
T5844 3194-3230 NN denotes GACGGGATGCAGAGGGGATCCGTCTGAGTCAGGCCC
T5847 3230-3231 HYPH denotes -
T5848 3231-3232 CD denotes 3
T5849 3232-3233 SYM denotes ′
T5850 3233-3235 , denotes ,
T5851 3235-3240 WDT denotes which
T5852 3241-3248 VBD denotes removed
T5853 3249-3252 DT denotes the
T5855 3253-3257 NN denotes stop
T5854 3258-3263 NN denotes codon
T5856 3264-3267 CC denotes and
T5857 3268-3278 VBD denotes introduced
T5858 3279-3280 DT denotes a
T5860 3281-3286 NN denotes BamHI
T5859 3287-3291 NN denotes site
T5861 3291-3293 , denotes ,
T5862 3293-3297 RB denotes then
T5863 3298-3302 IN denotes with
T5864 3303-3310 NNS denotes primers
T5866 3311-3312 CD denotes 5
T5867 3312-3313 SYM denotes ′
T5868 3313-3314 HYPH denotes -
T5865 3314-3348 NN denotes GACGGATCCCCTCTGAATTCCGTCCCCATCACCA
T5869 3348-3349 HYPH denotes -
T5870 3349-3350 CD denotes 3
T5871 3350-3351 SYM denotes ′
T5872 3352-3355 CC denotes and
T5873 3356-3357 CD denotes 5
T5875 3357-3358 SYM denotes ′
T5876 3358-3359 HYPH denotes -
T5874 3359-3393 NN denotes TGGTGATGGGGACGGAATACAGAGGGGATCCGTC
T5877 3393-3394 HYPH denotes -
T5878 3394-3395 CD denotes 3
T5879 3395-3396 SYM denotes ′
T5880 3396-3398 , denotes ,
T5881 3398-3403 WDT denotes which
T5882 3404-3414 VBD denotes introduced
T5883 3415-3417 DT denotes an
T5885 3418-3423 NN denotes EcoRI
T5884 3424-3428 NN denotes site
T5886 3428-3429 . denotes .
T5887 3429-3616 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 3430-3432 PRP denotes We
T5889 3433-3441 VBD denotes verified
T5890 3442-3445 DT denotes the
T5891 3446-3454 NN denotes sequence
T5892 3455-3459 IN denotes from
T5893 3460-3463 DT denotes the
T5895 3464-3471 VBN denotes mutated
T5894 3472-3479 NN denotes plasmid
T5896 3479-3481 , denotes ,
T5897 3481-3485 RB denotes then
T5898 3486-3494 VBD denotes digested
T5899 3495-3497 PRP denotes it
T5900 3498-3502 IN denotes with
T5901 3503-3508 NN denotes BamHI
T5902 3509-3512 CC denotes and
T5903 3513-3518 NN denotes EcoRI
T5904 3518-3520 , denotes ,
T5905 3520-3522 TO denotes to
T5906 3523-3529 VB denotes insert
T5907 3530-3532 IN denotes in
T5908 3533-3538 NN denotes frame
T5909 3539-3542 NN denotes GFP
T5910 3543-3552 NNS denotes sequences
T5911 3553-3557 IN denotes from
T5912 3558-3559 DT denotes a
T5914 3560-3563 NN denotes Bam
T5916 3564-3566 NN denotes HI
T5917 3566-3567 HYPH denotes -
T5915 3567-3572 NN denotes EcoRI
T5913 3573-3581 NN denotes fragment
T5918 3582-3584 IN denotes of
T5919 3585-3592 NN denotes plasmid
T5921 3593-3596 NN denotes phr
T5922 3596-3597 HYPH denotes -
T5920 3597-3600 NN denotes GFP
T5923 3600-3601 HYPH denotes -
T5924 3601-3602 CD denotes 1
T5925 3603-3604 -LRB- denotes (
T5926 3604-3614 NNP denotes Stratagene
T5927 3614-3615 -RRB- denotes )
T5928 3615-3616 . denotes .
T5929 3616-3859 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 3617-3619 PRP denotes We
T5931 3620-3628 VBD denotes verified
T5932 3629-3632 DT denotes the
T5933 3633-3641 NN denotes sequence
T5934 3642-3644 IN denotes of
T5935 3645-3649 DT denotes this
T5937 3650-3653 NN denotes p53
T5939 3653-3654 HYPH denotes -
T5938 3654-3657 NN denotes GFP
T5940 3658-3664 NN denotes fusion
T5936 3665-3673 NN denotes fragment
T5941 3673-3675 , denotes ,
T5942 3675-3679 RB denotes then
T5943 3680-3687 VBD denotes swapped
T5944 3688-3690 PRP denotes it
T5945 3691-3693 IN denotes in
T5946 3694-3697 DT denotes the
T5948 3698-3700 NN denotes L3
T5950 3700-3701 HYPH denotes -
T5951 3701-3717 NN denotes p53PmlEagPuroΔTK
T5952 3717-3718 HYPH denotes -
T5949 3718-3720 NN denotes 1L
T5947 3721-3728 NN denotes plasmid
T5953 3729-3730 -LRB- denotes (
T5954 3730-3733 VB denotes see
T5955 3734-3739 RB denotes above
T5956 3739-3740 -RRB- denotes )
T5957 3741-3743 IN denotes by
T5958 3744-3751 NN denotes HindIII
T5960 3752-3755 CC denotes and
T5961 3756-3760 NN denotes FseI
T5959 3761-3770 NN denotes digestion
T5962 3770-3772 , denotes ,
T5963 3772-3781 VBG denotes resulting
T5964 3782-3784 IN denotes in
T5965 3785-3788 DT denotes the
T5967 3789-3795 NN denotes p53GFP
T5968 3796-3804 NN denotes exchange
T5966 3805-3814 NN denotes construct
T5969 3814-3816 , denotes ,
T5970 3816-3819 DT denotes the
T5971 3820-3828 NN denotes sequence
T5972 3829-3834 WDT denotes which
T5974 3835-3838 VBD denotes was
T5973 3839-3847 VBN denotes verified
T5975 3848-3854 IN denotes before
T5976 3855-3858 NN denotes use
T5977 3858-3859 . denotes .
T5978 3859-4035 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 3860-3863 DT denotes The
T5981 3864-3872 NN denotes p53ΔPGFP
T5982 3873-3881 NN denotes exchange
T5980 3882-3891 NN denotes construct
T5984 3892-3895 VBD denotes was
T5983 3896-3906 VBN denotes engineered
T5985 3907-3909 IN denotes by
T5986 3910-3919 VBG denotes combining
T5987 3920-3929 NNS denotes sequences
T5988 3930-3934 IN denotes from
T5989 3935-3938 DT denotes the
T5991 3939-3945 NN denotes p53GFP
T5992 3946-3954 NN denotes exchange
T5990 3955-3962 NN denotes plasmid
T5993 3963-3966 CC denotes and
T5994 3967-3976 NNS denotes sequences
T5995 3977-3981 IN denotes from
T5996 3982-3985 DT denotes the
T5998 3986-3991 NN denotes p53ΔP
T5999 3992-4001 NN denotes targeting
T5997 4002-4011 NN denotes construct
T6000 4012-4021 VBN denotes described
T6001 4022-4030 RB denotes recently
T6002 4031-4032 -LRB- denotes (
T6003 4032-4033 CD denotes 5
T6004 4033-4034 -RRB- denotes )
T6005 4034-4035 . denotes .
T6006 4035-4078 sentence denotes Its sequence was also verified before use.
T6007 4036-4039 PRP$ denotes Its
T6008 4040-4048 NN denotes sequence
T6010 4049-4052 VBD denotes was
T6011 4053-4057 RB denotes also
T6009 4058-4066 VBN denotes verified
T6012 4067-4073 IN denotes before
T6013 4074-4077 NN denotes use
T6014 4077-4078 . denotes .
T6352 4080-4089 NNS denotes Sequences
T6353 4090-4093 CC denotes and
T6354 4094-4097 NN denotes use
T6355 4098-4100 IN denotes of
T6356 4101-4104 NN denotes PCR
T6357 4105-4112 NNS denotes primers
T6358 4112-4912 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 4113-4114 LS denotes a
T6361 4114-4116 : denotes :
T6362 4116-4117 CD denotes 5
T6363 4117-4118 SYM denotes ′
T6364 4118-4119 HYPH denotes -
T6360 4119-4143 NN denotes CCCCGGCCCTCACCCTCATCTTCG
T6366 4143-4144 HYPH denotes -
T6367 4144-4145 CD denotes 3
T6368 4145-4146 SYM denotes ′
T6369 4146-4148 , denotes ,
T6370 4148-4152 IN denotes from
T6371 4153-4156 DT denotes the
T6373 4157-4162 NN denotes PuΔTK
T6372 4163-4167 NN denotes gene
T6374 4167-4169 , denotes ,
T6375 4169-4175 NNS denotes assays
T6376 4176-4185 VBG denotes targeting
T6377 4186-4188 IN denotes of
T6378 4189-4193 NN denotes Flox
T6379 4194-4201 NN denotes plasmid
T6380 4201-4202 : denotes ;
T6381 4203-4204 LS denotes b
T6383 4204-4206 : denotes :
T6384 4206-4207 CD denotes 5
T6385 4207-4208 SYM denotes ′
T6386 4208-4209 HYPH denotes -
T6382 4209-4233 NN denotes AACAAAACAAAACAGCAGCAACAA
T6387 4233-4234 HYPH denotes -
T6388 4234-4235 CD denotes 3
T6389 4235-4236 SYM denotes ′
T6390 4236-4238 , denotes ,
T6391 4238-4242 IN denotes from
T6392 4243-4252 NNS denotes sequences
T6393 4253-4263 RB denotes downstream
T6395 4264-4266 IN denotes of
T6396 4267-4270 DT denotes the
T6398 4271-4274 NN denotes p53
T6397 4275-4279 NN denotes gene
T6399 4280-4283 CC denotes and
T6394 4284-4291 IN denotes outside
T6400 4292-4296 NN denotes Flox
T6401 4297-4306 NNS denotes sequences
T6402 4306-4308 , denotes ,
T6403 4308-4314 NNS denotes assays
T6404 4315-4324 VBG denotes targeting
T6405 4325-4327 IN denotes of
T6406 4328-4332 NN denotes Flox
T6407 4333-4336 CC denotes and
T6408 4337-4341 NN denotes RMCE
T6409 4342-4346 IN denotes with
T6410 4347-4353 NN denotes p53GFP
T6412 4354-4356 CC denotes or
T6413 4357-4365 NN denotes p53ΔPGFP
T6411 4366-4374 NNS denotes plasmids
T6414 4374-4375 : denotes ;
T6415 4376-4377 LS denotes c
T6417 4377-4379 : denotes :
T6418 4379-4380 CD denotes 5
T6419 4380-4381 SYM denotes ′
T6420 4381-4382 HYPH denotes -
T6416 4382-4407 NN denotes TGAAGAGCAAGGGCGTGGTGAAGGA
T6421 4407-4408 HYPH denotes -
T6422 4408-4409 CD denotes 3
T6423 4409-4410 SYM denotes ′
T6424 4410-4412 , denotes ,
T6425 4412-4416 IN denotes from
T6426 4417-4420 NN denotes GFP
T6427 4421-4430 NNS denotes sequences
T6428 4430-4432 , denotes ,
T6429 4432-4438 NNS denotes assays
T6430 4439-4443 NN denotes RMCE
T6431 4444-4448 IN denotes with
T6432 4449-4455 NN denotes p53GFP
T6433 4456-4466 NN denotes orp53ΔPGFP
T6434 4467-4475 NNS denotes plasmids
T6435 4475-4476 : denotes ;
T6436 4477-4478 LS denotes d
T6438 4478-4480 : denotes :
T6439 4480-4481 CD denotes 5
T6440 4481-4482 SYM denotes ′
T6441 4482-4483 HYPH denotes -
T6437 4483-4511 NN denotes CAAAAAATGGAAGGAAATCAGGAACTAA
T6442 4511-4512 HYPH denotes -
T6443 4512-4513 CD denotes 3
T6444 4513-4514 SYM denotes ′
T6445 4514-4516 , denotes ,
T6446 4516-4520 IN denotes from
T6447 4521-4524 NN denotes p53
T6448 4525-4531 NN denotes intron
T6449 4532-4533 CD denotes 3
T6450 4533-4535 , denotes ,
T6451 4535-4538 CC denotes and
T6452 4539-4540 LS denotes e
T6454 4540-4542 : denotes :
T6455 4542-4543 CD denotes 5
T6456 4543-4544 SYM denotes ′
T6457 4544-4545 HYPH denotes -
T6453 4545-4570 NN denotes TCTAGACAGAGAAAAAAGAGGCATT
T6458 4570-4571 HYPH denotes -
T6459 4571-4572 CD denotes 3
T6460 4572-4573 SYM denotes ′
T6461 4573-4575 , denotes ,
T6462 4575-4579 IN denotes from
T6463 4580-4583 NN denotes p53
T6464 4584-4590 NN denotes intron
T6465 4591-4592 CD denotes 4
T6466 4592-4594 , denotes ,
T6467 4594-4599 NN denotes assay
T6468 4600-4604 NN denotes RMCE
T6469 4605-4609 IN denotes with
T6470 4610-4618 NN denotes p53ΔPGFP
T6471 4619-4626 NN denotes plasmid
T6472 4626-4627 : denotes ;
T6473 4628-4629 LS denotes f
T6475 4629-4631 : denotes :
T6476 4631-4632 CD denotes 5
T6477 4632-4633 SYM denotes ′
T6478 4633-4634 HYPH denotes -
T6474 4634-4658 NN denotes ATGGGAGGCTGCCAGTCCTAACCC
T6479 4659-4662 CC denotes and
T6480 4663-4664 LS denotes g
T6482 4664-4666 : denotes :
T6483 4666-4667 CD denotes 5
T6484 4667-4668 SYM denotes ′
T6485 4668-4669 HYPH denotes -
T6481 4669-4695 NN denotes GTGTTTCATTAGTTCCCCACCTTGAC
T6486 4695-4696 HYPH denotes -
T6487 4696-4697 CD denotes 3
T6488 4697-4698 SYM denotes ′
T6365 4699-4706 VBP denotes amplify
T6490 4707-4710 DT denotes the
T6492 4711-4713 NN denotes WT
T6493 4714-4717 NN denotes p53
T6491 4718-4724 NN denotes allele
T6494 4725-4734 IN denotes according
T6495 4735-4737 IN denotes to
T6496 4738-4745 NNP denotes Taconic
T6498 4745-4747 POS denotes 's
T6497 4748-4758 NNS denotes procedures
T6499 4758-4760 , denotes ,
T6500 4760-4761 LS denotes h
T6502 4761-4763 : denotes :
T6503 4763-4764 CD denotes 5
T6504 4764-4765 SYM denotes ′
T6505 4765-4766 HYPH denotes -
T6501 4766-4791 NN denotes TTTACGGAGCCCTGGCGCTCGATGT
T6506 4791-4792 HYPH denotes -
T6507 4792-4793 CD denotes 3
T6508 4793-4794 SYM denotes ′
T6509 4795-4798 CC denotes and
T6510 4799-4800 LS denotes i
T6512 4800-4802 : denotes :
T6513 4802-4803 CD denotes 5
T6514 4803-4804 SYM denotes ′
T6515 4804-4805 HYPH denotes -
T6511 4805-4830 NN denotes GTGGGAGGGACAAAAGTTCGAGGCC
T6516 4830-4831 HYPH denotes -
T6517 4831-4832 CD denotes 3
T6518 4832-4833 SYM denotes ′
T6489 4834-4841 VBP denotes amplify
T6519 4842-4845 DT denotes the
T6521 4846-4849 NN denotes Neo
T6520 4850-4856 NN denotes marker
T6522 4857-4859 IN denotes in
T6523 4860-4863 DT denotes the
T6525 4864-4867 NN denotes p53
T6526 4868-4870 NN denotes KO
T6524 4871-4877 NN denotes allele
T6527 4878-4887 IN denotes according
T6528 4888-4890 IN denotes to
T6529 4891-4898 NNP denotes Taconic
T6531 4898-4900 POS denotes 's
T6530 4901-4911 NNS denotes procedures
T6532 4911-4912 . denotes .
T6727 4914-4918 NN denotes Cell
T6728 4919-4926 NN denotes culture
T6729 4927-4937 NNS denotes conditions
T6730 4937-5065 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 4938-4945 JJ denotes Primary
T6732 4946-4950 NNS denotes MEFs
T6734 4950-4952 , denotes ,
T6735 4952-4960 VBN denotes isolated
T6736 4961-4965 IN denotes from
T6737 4966-4970 CD denotes 13.5
T6738 4971-4974 NN denotes day
T6739 4975-4982 NNS denotes embryos
T6740 4982-4984 , denotes ,
T6741 4984-4988 VBD denotes were
T6733 4989-4997 VBN denotes cultured
T6742 4998-5000 IN denotes in
T6743 5001-5005 NN denotes DMEM
T6744 5006-5010 IN denotes with
T6745 5011-5013 CD denotes 15
T6746 5013-5014 NN denotes %
T6747 5015-5018 NN denotes FBS
T6748 5018-5020 , denotes ,
T6749 5020-5023 CD denotes 100
T6750 5024-5026 NN denotes mM
T6751 5027-5030 NN denotes BME
T6752 5030-5032 , denotes ,
T6753 5032-5033 CD denotes 2
T6754 5034-5036 NN denotes mM
T6756 5037-5038 NN denotes l
T6757 5038-5039 HYPH denotes -
T6755 5039-5048 NN denotes glutamine
T6758 5049-5052 CC denotes and
T6759 5053-5064 NNS denotes antibiotics
T6760 5064-5065 . denotes .
T6761 5065-5241 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 5066-5069 CD denotes 129
T6764 5069-5070 HYPH denotes /
T6763 5070-5075 NN denotes SvJae
T6766 5076-5078 NN denotes ES
T6765 5079-5084 NNS denotes cells
T6768 5085-5089 VBD denotes were
T6767 5090-5095 VBN denotes grown
T6769 5096-5098 IN denotes in
T6770 5099-5102 DT denotes the
T6772 5103-5107 JJ denotes same
T6771 5108-5114 NN denotes medium
T6773 5115-5127 VBN denotes supplemented
T6774 5128-5132 IN denotes with
T6775 5133-5137 CD denotes 1000
T6776 5138-5139 NN denotes U
T6778 5139-5140 SYM denotes /
T6779 5140-5142 NN denotes ml
T6777 5143-5148 NN denotes ESGRO
T6780 5149-5150 -LRB- denotes (
T6781 5150-5158 NNP denotes Chemicon
T6782 5158-5159 -RRB- denotes )
T6783 5159-5161 , denotes ,
T6784 5161-5163 IN denotes on
T6785 5164-5165 DT denotes a
T6786 5166-5171 NN denotes layer
T6787 5172-5174 IN denotes of
T6788 5175-5184 NN denotes mitomycin
T6789 5185-5186 NN denotes C
T6791 5186-5187 HYPH denotes -
T6790 5187-5194 JJ denotes treated
T6793 5195-5202 NN denotes SNLPuro
T6794 5202-5203 HYPH denotes -
T6795 5203-5204 CD denotes 7
T6796 5204-5205 HYPH denotes /
T6797 5205-5206 CD denotes 4
T6792 5207-5214 NNS denotes feeders
T6798 5215-5216 -LRB- denotes (
T6800 5216-5220 JJ denotes kind
T6799 5221-5225 NN denotes gift
T6801 5226-5228 IN denotes of
T6802 5229-5231 NNP denotes A.
T6803 5232-5239 NNP denotes Bradley
T6804 5239-5240 -RRB- denotes )
T6805 5240-5241 . denotes .
T6806 5241-5324 sentence denotes Selections were performed with 2 μg/ml puromycin, 0.2 μM FIAU or 2 μM ganciclovir.
T6807 5242-5252 NNS denotes Selections
T6809 5253-5257 VBD denotes were
T6808 5258-5267 VBN denotes performed
T6810 5268-5272 IN denotes with
T6811 5273-5274 CD denotes 2
T6812 5275-5277 NN denotes μg
T6814 5277-5278 SYM denotes /
T6815 5278-5280 NN denotes ml
T6813 5281-5290 NN denotes puromycin
T6816 5290-5292 , denotes ,
T6817 5292-5295 CD denotes 0.2
T6818 5296-5298 NN denotes μM
T6819 5299-5303 NN denotes FIAU
T6820 5304-5306 CC denotes or
T6821 5307-5308 CD denotes 2
T6822 5309-5311 NN denotes μM
T6823 5312-5323 NN denotes ganciclovir
T6824 5323-5324 . denotes .
T6929 5326-5335 NN denotes Targeting
T6931 5335-5336 HYPH denotes /
T6930 5336-5346 NN denotes genotyping
T6932 5347-5349 IN denotes of
T6933 5350-5353 DT denotes the
T6935 5354-5358 JJ denotes RMCE
T6937 5358-5359 HYPH denotes -
T6936 5359-5364 JJ denotes ready
T6934 5365-5370 NN denotes locus
T6938 5370-5524 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 5371-5373 CD denotes 29
T6941 5373-5374 HYPH denotes /
T6940 5374-5379 NN denotes SvJae
T6943 5380-5382 NN denotes ES
T6942 5383-5388 NNS denotes cells
T6945 5389-5393 VBD denotes were
T6944 5394-5408 VBN denotes electroporated
T6946 5409-5413 IN denotes with
T6947 5414-5417 DT denotes the
T6949 5418-5422 NN denotes Flox
T6948 5423-5432 NN denotes construct
T6950 5433-5443 VBN denotes linearized
T6951 5444-5448 IN denotes with
T6952 5449-5453 NN denotes PmeI
T6953 5453-5455 , denotes ,
T6954 5455-5458 CC denotes and
T6955 5459-5468 NN denotes puromycin
T6956 5469-5478 JJ denotes resistant
T6957 5479-5485 NNS denotes clones
T6959 5486-5490 VBD denotes were
T6958 5491-5499 VBN denotes analyzed
T6960 5500-5502 IN denotes as
T6961 5503-5512 VBN denotes described
T6962 5513-5514 -LRB- denotes (
T6963 5514-5520 NN denotes Figure
T6964 5521-5522 CD denotes 2
T6965 5522-5523 -RRB- denotes )
T6966 5523-5524 . denotes .
T6967 5524-5604 sentence denotes Two clones were injected into blastocysts and transmitted through the germline.
T6968 5525-5528 CD denotes Two
T6969 5529-5535 NNS denotes clones
T6971 5536-5540 VBD denotes were
T6970 5541-5549 VBN denotes injected
T6972 5550-5554 IN denotes into
T6973 5555-5566 NNS denotes blastocysts
T6974 5567-5570 CC denotes and
T6975 5571-5582 VBN denotes transmitted
T6976 5583-5590 IN denotes through
T6977 5591-5594 DT denotes the
T6978 5595-5603 NN denotes germline
T6979 5603-5604 . denotes .
T7192 5606-5616 VBG denotes Performing
T7193 5617-5621 NN denotes RMCE
T7194 5622-5624 IN denotes in
T7195 5625-5627 NN denotes ES
T7196 5628-5633 NNS denotes cells
T7197 5633-5848 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 5634-5635 DT denotes A
T7199 5636-5641 NN denotes total
T7201 5642-5644 IN denotes of
T7202 5645-5646 CD denotes 8
T7204 5647-5648 HYPH denotes ×
T7203 5649-5652 CD denotes 105
T7206 5653-5660 NN denotes p53RMCE
T7207 5660-5661 HYPH denotes /
T7208 5661-5662 SYM denotes +
T7209 5663-5665 NN denotes ES
T7205 5666-5671 NNS denotes cells
T7210 5672-5676 VBD denotes were
T7200 5677-5682 VBN denotes grown
T7211 5683-5690 IN denotes without
T7212 5691-5700 NN denotes puromycin
T7213 5701-5704 IN denotes for
T7214 5705-5707 CD denotes 12
T7215 5708-5709 NN denotes h
T7216 5709-5711 , denotes ,
T7217 5711-5725 VBN denotes electroporated
T7218 5726-5730 IN denotes with
T7219 5731-5733 CD denotes 15
T7221 5734-5736 NN denotes μg
T7222 5737-5740 NN denotes CMV
T7224 5740-5741 HYPH denotes -
T7223 5741-5744 NN denotes Cre
T7220 5745-5752 NN denotes plasmid
T7225 5753-5754 -LRB- denotes (
T7226 5754-5760 NN denotes pOG231
T7227 5760-5761 -RRB- denotes )
T7228 5762-5765 CC denotes and
T7229 5766-5769 CD denotes 200
T7230 5770-5772 NN denotes μg
T7231 5773-5775 IN denotes of
T7232 5776-5779 DT denotes the
T7234 5780-5788 NN denotes exchange
T7233 5789-5798 NN denotes construct
T7235 5798-5800 , denotes ,
T7236 5800-5803 CC denotes and
T7237 5804-5810 VBN denotes plated
T7238 5811-5813 IN denotes in
T7239 5814-5817 NN denotes T25
T7240 5818-5824 NNS denotes flasks
T7241 5825-5827 IN denotes at
T7242 5828-5831 CD denotes 105
T7243 5832-5837 NNS denotes cells
T7244 5838-5841 IN denotes per
T7245 5842-5847 NN denotes flask
T7246 5847-5848 . denotes .
T7247 5848-5909 sentence denotes FIAU was added to the medium 3–4 days after electroporation.
T7248 5849-5853 NN denotes FIAU
T7250 5854-5857 VBD denotes was
T7249 5858-5863 VBN denotes added
T7251 5864-5866 IN denotes to
T7252 5867-5870 DT denotes the
T7253 5871-5877 NN denotes medium
T7254 5878-5879 CD denotes 3
T7256 5879-5880 HYPH denotes –
T7255 5880-5881 CD denotes 4
T7257 5882-5886 NNS denotes days
T7258 5887-5892 IN denotes after
T7259 5893-5908 NN denotes electroporation
T7260 5908-5909 . denotes .
T7261 5909-6088 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 5910-5920 JJ denotes Individual
T7263 5921-5927 NNS denotes clones
T7265 5927-5929 , denotes ,
T7266 5929-5935 VBN denotes picked
T7267 5936-5938 CD denotes 10
T7268 5939-5943 NNS denotes days
T7269 5944-5949 IN denotes after
T7270 5950-5965 NN denotes electroporation
T7271 5965-5967 , denotes ,
T7272 5967-5971 VBD denotes were
T7264 5972-5977 VBN denotes grown
T7273 5978-5980 IN denotes in
T7274 5981-5983 CD denotes 96
T7276 5983-5984 HYPH denotes -
T7275 5984-5988 NN denotes well
T7277 5989-5995 NNS denotes plates
T7278 5996-5999 CC denotes and
T7279 6000-6008 VBN denotes expanded
T7280 6009-6011 TO denotes to
T7281 6012-6020 VB denotes generate
T7282 6021-6030 JJ denotes duplicate
T7283 6031-6037 NNS denotes plates
T7284 6038-6041 IN denotes for
T7285 6042-6050 VBG denotes freezing
T7286 6051-6054 CC denotes and
T7287 6055-6058 NN denotes DNA
T7288 6059-6067 NN denotes analysis
T7289 6068-6070 IN denotes by
T7290 6071-6074 NN denotes PCR
T7291 6075-6078 CC denotes and
T7292 6079-6087 NNP denotes Southern
T7293 6087-6088 . denotes .
T7497 6090-6100 VBG denotes Performing
T7498 6101-6105 NN denotes RMCE
T7499 6106-6108 IN denotes in
T7500 6109-6113 NNS denotes MEFs
T7501 6113-6354 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 6114-6115 DT denotes A
T7503 6116-6121 NN denotes total
T7505 6122-6124 IN denotes of
T7506 6125-6128 CD denotes 106
T7508 6129-6136 NN denotes p53RMCE
T7510 6136-6137 HYPH denotes /
T7511 6137-6138 SYM denotes −
T7509 6139-6143 NNS denotes MEFs
T7507 6144-6149 NNS denotes cells
T7512 6150-6154 VBD denotes were
T7504 6155-6160 VBN denotes grown
T7513 6161-6168 IN denotes without
T7514 6169-6178 NN denotes puromycin
T7515 6179-6182 IN denotes for
T7516 6183-6185 CD denotes 12
T7517 6186-6187 NN denotes h
T7518 6187-6189 , denotes ,
T7519 6189-6203 VBN denotes electroporated
T7520 6204-6208 IN denotes with
T7521 6209-6210 CD denotes 3
T7522 6211-6213 NN denotes μg
T7523 6214-6220 NN denotes pOG231
T7524 6221-6224 CC denotes and
T7525 6225-6227 CD denotes 30
T7526 6228-6230 NN denotes μg
T7528 6231-6239 NN denotes exchange
T7527 6240-6249 NN denotes construct
T7529 6249-6251 , denotes ,
T7530 6251-6254 CC denotes and
T7531 6255-6261 VBN denotes plated
T7532 6262-6264 IN denotes in
T7533 6265-6266 DT denotes a
T7535 6267-6273 JJ denotes single
T7536 6274-6276 CD denotes 10
T7537 6277-6279 NN denotes cm
T7538 6279-6280 HYPH denotes -
T7534 6280-6284 NN denotes dish
T7539 6284-6286 , denotes ,
T7540 6286-6291 VBN denotes grown
T7541 6292-6295 IN denotes for
T7542 6296-6297 CD denotes 3
T7543 6298-6302 NNS denotes days
T7544 6303-6307 RB denotes then
T7545 6308-6313 VBN denotes split
T7546 6314-6316 IN denotes in
T7547 6317-6324 JJ denotes several
T7548 6325-6331 NNS denotes dishes
T7549 6332-6334 IN denotes at
T7550 6335-6338 CD denotes 105
T7551 6339-6344 NNS denotes cells
T7552 6345-6348 IN denotes per
T7553 6349-6353 NN denotes dish
T7554 6353-6354 . denotes .
T7555 6354-6442 sentence denotes FIAU or ganciclovir was added to the medium 4 days after electroporation, for 3–4 days.
T7556 6355-6359 NN denotes FIAU
T7558 6360-6362 CC denotes or
T7559 6363-6374 NN denotes ganciclovir
T7560 6375-6378 VBD denotes was
T7557 6379-6384 VBN denotes added
T7561 6385-6387 IN denotes to
T7562 6388-6391 DT denotes the
T7563 6392-6398 NN denotes medium
T7564 6399-6400 CD denotes 4
T7565 6401-6405 NNS denotes days
T7566 6406-6411 IN denotes after
T7567 6412-6427 NN denotes electroporation
T7568 6427-6429 , denotes ,
T7569 6429-6432 IN denotes for
T7570 6433-6434 CD denotes 3
T7572 6434-6435 SYM denotes –
T7571 6435-6436 CD denotes 4
T7573 6437-6441 NNS denotes days
T7574 6441-6442 . denotes .
T7575 6442-6561 sentence denotes Clones, picked 10 days after electroporation, were grown in 24-well plates and expanded for freezing and DNA analysis.
T7576 6443-6449 NNS denotes Clones
T7578 6449-6451 , denotes ,
T7579 6451-6457 VBN denotes picked
T7580 6458-6460 CD denotes 10
T7581 6461-6465 NNS denotes days
T7582 6466-6471 IN denotes after
T7583 6472-6487 NN denotes electroporation
T7584 6487-6489 , denotes ,
T7585 6489-6493 VBD denotes were
T7577 6494-6499 VBN denotes grown
T7586 6500-6502 IN denotes in
T7587 6503-6505 CD denotes 24
T7589 6505-6506 HYPH denotes -
T7588 6506-6510 NN denotes well
T7590 6511-6517 NNS denotes plates
T7591 6518-6521 CC denotes and
T7592 6522-6530 VBN denotes expanded
T7593 6531-6534 IN denotes for
T7594 6535-6543 NN denotes freezing
T7595 6544-6547 CC denotes and
T7596 6548-6551 NN denotes DNA
T7597 6552-6560 NN denotes analysis
T7598 6560-6561 . denotes .
T8070 6563-6570 NNP denotes Western
T8072 6570-6571 HYPH denotes -
T8071 6571-6576 NNS denotes blots
T8073 6576-6873 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 6577-6582 NNS denotes Cells
T8076 6582-6584 , denotes ,
T8077 6584-6593 JJ denotes untreated
T8078 6594-6596 CC denotes or
T8079 6597-6604 VBN denotes treated
T8080 6605-6608 IN denotes for
T8081 6609-6611 CD denotes 24
T8082 6612-6613 NN denotes h
T8083 6614-6618 IN denotes with
T8084 6619-6622 CD denotes 0.5
T8085 6623-6625 NN denotes μg
T8087 6625-6626 SYM denotes /
T8088 6626-6628 NN denotes ml
T8086 6629-6639 NN denotes adriamycin
T8089 6639-6641 , denotes ,
T8090 6641-6645 VBD denotes were
T8075 6646-6651 VBN denotes lysed
T8091 6652-6654 IN denotes on
T8092 6655-6658 DT denotes the
T8093 6659-6663 NN denotes dish
T8094 6664-6666 IN denotes in
T8095 6667-6668 DT denotes a
T8096 6669-6675 NN denotes buffer
T8097 6676-6686 VBG denotes consisting
T8098 6687-6689 IN denotes of
T8099 6690-6692 CD denotes 50
T8100 6693-6695 NN denotes mM
T8101 6696-6700 NN denotes Tris
T8102 6701-6702 -LRB- denotes (
T8103 6702-6704 NN denotes pH
T8104 6705-6708 CD denotes 8.0
T8105 6708-6709 -RRB- denotes )
T8106 6709-6711 , denotes ,
T8107 6711-6712 CD denotes 5
T8108 6713-6715 NN denotes mM
T8109 6716-6720 NN denotes EDTA
T8110 6720-6722 , denotes ,
T8111 6722-6725 CD denotes 150
T8112 6726-6728 NN denotes mM
T8113 6729-6733 NN denotes NaCl
T8114 6733-6735 , denotes ,
T8115 6735-6738 CD denotes 0.5
T8116 6738-6739 NN denotes %
T8118 6740-6747 NN denotes Nonidet
T8119 6748-6749 NN denotes P
T8120 6749-6750 HYPH denotes -
T8117 6750-6752 NN denotes 40
T8121 6752-6754 , denotes ,
T8122 6754-6755 CD denotes 1
T8123 6756-6758 NN denotes mM
T8124 6759-6763 NN denotes PMSF
T8125 6763-6765 , denotes ,
T8126 6765-6766 CD denotes 1
T8127 6767-6769 NN denotes mM
T8129 6770-6776 NN denotes sodium
T8128 6777-6785 NN denotes vanadate
T8130 6785-6787 , denotes ,
T8131 6787-6789 CD denotes 10
T8132 6790-6792 NN denotes mM
T8133 6793-6796 NNP denotes NaF
T8134 6797-6800 CC denotes and
T8135 6801-6809 NN denotes Complete
T8136 6810-6814 NN denotes Mini
T8138 6815-6823 NN denotes Protease
T8137 6824-6834 NNS denotes Inhibitors
T8139 6835-6836 -LRB- denotes (
T8141 6836-6841 NNP denotes Roche
T8140 6842-6853 NNP denotes Diagnostics
T8142 6853-6854 -RRB- denotes )
T8143 6855-6857 IN denotes at
T8144 6858-6859 CD denotes 4
T8145 6859-6861 NNS denotes °C
T8146 6862-6865 IN denotes for
T8147 6866-6868 CD denotes 30
T8148 6869-6872 NN denotes min
T8149 6872-6873 . denotes .
T8150 6873-6935 sentence denotes Lysates were scraped, then spun at 6000× g at 4°C for 10 min.
T8151 6874-6881 NNS denotes Lysates
T8153 6882-6886 VBD denotes were
T8152 6887-6894 VBN denotes scraped
T8154 6894-6896 , denotes ,
T8155 6896-6900 RB denotes then
T8156 6901-6905 VBN denotes spun
T8157 6906-6908 IN denotes at
T8158 6909-6913 CD denotes 6000
T8160 6913-6914 SYM denotes ×
T8159 6915-6916 NN denotes g
T8161 6917-6919 IN denotes at
T8162 6920-6921 CD denotes 4
T8163 6921-6923 NNS denotes °C
T8164 6924-6927 IN denotes for
T8165 6928-6930 CD denotes 10
T8166 6931-6934 NN denotes min
T8167 6934-6935 . denotes .
T8168 6935-7027 sentence denotes Protein concentration in the supernatant was determined using the Bio-Rad DC protein assay.
T8169 6936-6943 NN denotes Protein
T8170 6944-6957 NN denotes concentration
T8172 6958-6960 IN denotes in
T8173 6961-6964 DT denotes the
T8174 6965-6976 NN denotes supernatant
T8175 6977-6980 VBD denotes was
T8171 6981-6991 VBN denotes determined
T8176 6992-6997 VBG denotes using
T8177 6998-7001 DT denotes the
T8179 7002-7005 NN denotes Bio
T8181 7005-7006 HYPH denotes -
T8180 7006-7009 NN denotes Rad
T8182 7010-7012 NN denotes DC
T8183 7013-7020 NN denotes protein
T8178 7021-7026 NN denotes assay
T8184 7026-7027 . denotes .
T8185 7027-7182 sentence denotes Lysates were separated on single percentage SDS/PAGE gels, then electrophoretically transferred to poly(vinylidene difluoride), using standard procedures.
T8186 7028-7035 NNS denotes Lysates
T8188 7036-7040 VBD denotes were
T8187 7041-7050 VBN denotes separated
T8189 7051-7053 IN denotes on
T8190 7054-7060 JJ denotes single
T8191 7061-7071 NN denotes percentage
T8193 7072-7075 NN denotes SDS
T8195 7075-7076 HYPH denotes /
T8194 7076-7080 NN denotes PAGE
T8192 7081-7085 NNS denotes gels
T8196 7085-7087 , denotes ,
T8197 7087-7091 RB denotes then
T8199 7092-7111 RB denotes electrophoretically
T8198 7112-7123 VBN denotes transferred
T8200 7124-7126 IN denotes to
T8201 7127-7131 NN denotes poly
T8202 7131-7132 -LRB- denotes (
T8204 7132-7142 NN denotes vinylidene
T8203 7143-7153 NN denotes difluoride
T8205 7153-7154 -RRB- denotes )
T8206 7154-7156 , denotes ,
T8207 7156-7161 VBG denotes using
T8208 7162-7170 JJ denotes standard
T8209 7171-7181 NNS denotes procedures
T8210 7181-7182 . denotes .
T8211 7182-7187 NNS denotes Blots
T8213 7182-7401 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 7188-7192 VBD denotes were
T8212 7193-7202 VBN denotes incubated
T8215 7203-7205 IN denotes in
T8216 7206-7207 CD denotes 5
T8217 7207-7208 NN denotes %
T8219 7209-7216 JJ denotes non-fat
T8220 7217-7222 VBN denotes dried
T8218 7223-7227 NN denotes milk
T8221 7228-7230 IN denotes in
T8222 7231-7235 NN denotes TBST
T8223 7236-7237 -LRB- denotes (
T8225 7237-7241 CD denotes 0.02
T8226 7242-7243 NN denotes M
T8227 7244-7248 NN denotes Tris
T8228 7248-7250 , denotes ,
T8229 7250-7252 NN denotes pH
T8230 7253-7256 CD denotes 7.6
T8231 7256-7257 HYPH denotes /
T8232 7257-7261 CD denotes 0.35
T8233 7262-7263 NN denotes M
T8234 7264-7268 NN denotes NaCl
T8235 7268-7269 HYPH denotes /
T8236 7269-7272 CD denotes 0.1
T8237 7272-7273 NN denotes %
T8224 7274-7279 NN denotes Tween
T8238 7279-7280 HYPH denotes -
T8239 7280-7282 CD denotes 20
T8240 7282-7283 -RRB- denotes )
T8241 7284-7287 IN denotes for
T8242 7288-7289 CD denotes 1
T8243 7290-7291 NN denotes h
T8244 7292-7294 IN denotes at
T8245 7295-7299 NN denotes room
T8246 7300-7311 NN denotes temperature
T8247 7312-7318 IN denotes before
T8248 7319-7326 VBG denotes probing
T8249 7327-7331 IN denotes with
T8250 7332-7339 JJ denotes primary
T8251 7340-7350 NNS denotes antibodies
T8252 7351-7358 IN denotes against
T8253 7359-7362 NN denotes p53
T8254 7363-7364 -LRB- denotes (
T8256 7364-7366 NN denotes CM
T8257 7366-7367 HYPH denotes -
T8258 7367-7368 CD denotes 5
T8259 7368-7370 , denotes ,
T8255 7370-7380 NNP denotes Novacastra
T8260 7380-7381 -RRB- denotes )
T8261 7382-7385 CC denotes and
T8262 7386-7387 HYPH denotes -
T8263 7387-7392 NN denotes actin
T8264 7393-7394 -LRB- denotes (
T8265 7394-7399 NNP denotes Sigma
T8266 7399-7400 -RRB- denotes )
T8267 7400-7401 . denotes .
T8268 7401-7507 sentence denotes Secondary antibodies used include peroxidase-conjugated goat anti-mouse IgG and anti-rabbit IgG (Pierce).
T8269 7402-7411 JJ denotes Secondary
T8270 7412-7422 NNS denotes antibodies
T8272 7423-7427 VBN denotes used
T8271 7428-7435 VBP denotes include
T8273 7436-7446 NN denotes peroxidase
T8275 7446-7447 HYPH denotes -
T8274 7447-7457 VBN denotes conjugated
T8277 7458-7462 NN denotes goat
T8278 7463-7473 NN denotes anti-mouse
T8276 7474-7477 NN denotes IgG
T8279 7478-7481 CC denotes and
T8280 7482-7493 NN denotes anti-rabbit
T8281 7494-7497 NN denotes IgG
T8282 7498-7499 -LRB- denotes (
T8283 7499-7505 NNP denotes Pierce
T8284 7505-7506 -RRB- denotes )
T8285 7506-7507 . denotes .
T8286 7507-7624 sentence denotes Probed blots were incubated with Pierce Supersignal West Pico chemiluminescent substrate and exposed to X-ray films.
T8287 7508-7514 VBN denotes Probed
T8288 7515-7520 NNS denotes blots
T8290 7521-7525 VBD denotes were
T8289 7526-7535 VBN denotes incubated
T8291 7536-7540 IN denotes with
T8292 7541-7547 NNP denotes Pierce
T8294 7548-7559 NNP denotes Supersignal
T8295 7560-7564 NNP denotes West
T8296 7565-7569 NNP denotes Pico
T8297 7570-7586 JJ denotes chemiluminescent
T8293 7587-7596 NN denotes substrate
T8298 7597-7600 CC denotes and
T8299 7601-7608 VBN denotes exposed
T8300 7609-7611 IN denotes to
T8301 7612-7617 NN denotes X-ray
T8302 7618-7623 NNS denotes films
T8303 7623-7624 . denotes .
T8467 7626-7630 NN denotes Flow
T8468 7631-7640 NN denotes cytometry
T8469 7640-7751 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 7641-7644 NN denotes Log
T8472 7645-7650 NN denotes phase
T8471 7651-7656 NNS denotes cells
T8474 7657-7661 VBD denotes were
T8473 7662-7672 VBN denotes irradiated
T8475 7673-7675 IN denotes at
T8476 7676-7678 NN denotes RT
T8477 7679-7683 IN denotes with
T8478 7684-7685 DT denotes a
T8480 7686-7688 CD denotes 60
T8482 7689-7691 NN denotes Co
T8481 7692-7693 NN denotes γ
T8483 7693-7694 SYM denotes -
T8479 7694-7704 NN denotes irradiator
T8484 7705-7707 IN denotes at
T8485 7708-7713 NNS denotes doses
T8486 7714-7716 IN denotes of
T8487 7717-7718 CD denotes 6
T8489 7719-7721 CC denotes or
T8490 7722-7724 CD denotes 12
T8488 7725-7727 NN denotes Gy
T8491 7728-7731 CC denotes and
T8492 7732-7741 VBN denotes incubated
T8493 7742-7745 IN denotes for
T8494 7746-7748 CD denotes 24
T8495 7749-7750 NN denotes h
T8496 7750-7751 . denotes .
T8497 7751-7944 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 7752-7757 NNS denotes Cells
T8500 7758-7762 VBD denotes were
T8501 7763-7767 RB denotes then
T8502 7768-7773 NN denotes pulse
T8503 7773-7774 HYPH denotes -
T8499 7774-7781 VBN denotes labeled
T8504 7782-7785 IN denotes for
T8505 7786-7787 CD denotes 1
T8506 7788-7789 NN denotes h
T8507 7790-7794 IN denotes with
T8508 7795-7799 NN denotes BrdU
T8509 7800-7801 -LRB- denotes (
T8511 7801-7803 CD denotes 10
T8510 7804-7806 NN denotes μM
T8512 7806-7807 -RRB- denotes )
T8513 7807-7809 , denotes ,
T8514 7809-7814 VBN denotes fixed
T8515 7815-7817 IN denotes in
T8516 7818-7820 CD denotes 70
T8517 7820-7821 NN denotes %
T8518 7822-7829 NN denotes ethanol
T8519 7829-7831 , denotes ,
T8520 7831-7837 JJ denotes double
T8522 7837-7838 HYPH denotes -
T8521 7838-7845 VBN denotes stained
T8523 7846-7850 IN denotes with
T8524 7851-7855 NN denotes FITC
T8525 7856-7865 NN denotes anti-BrdU
T8526 7866-7869 CC denotes and
T8527 7870-7879 NN denotes propidium
T8528 7880-7886 NN denotes iodide
T8529 7886-7888 , denotes ,
T8530 7888-7892 RB denotes then
T8531 7893-7899 VBN denotes sorted
T8532 7900-7902 IN denotes by
T8533 7903-7908 VBG denotes using
T8534 7909-7910 DT denotes a
T8536 7911-7917 NNP denotes Becton
T8537 7918-7927 NNP denotes Dickinson
T8538 7928-7935 NNP denotes FACScan
T8535 7936-7943 NN denotes machine
T8539 7943-7944 . denotes .
T8540 7944-8001 sentence denotes Data were analyzed using Becton Dickinson Cellquest Pro.
T8541 7945-7949 NNS denotes Data
T8543 7950-7954 VBD denotes were
T8542 7955-7963 VBN denotes analyzed
T8544 7964-7969 VBG denotes using
T8545 7970-7976 NNP denotes Becton
T8546 7977-7986 NNP denotes Dickinson
T8548 7987-7996 NNP denotes Cellquest
T8547 7997-8000 NNP denotes Pro
T8549 8000-8001 . denotes .
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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