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create E2 knockout mice, we used gene targeting in mouse ES cells to disrupt the E2 gene. The overall strategy for disrupting the E2 gene is illustrated in Fig. 1A. The gene targeting construct was designed to replace a 1.67 kb EcoRV/Smal genomic DNA fragment encompassing part of Exon 4 and all of Exon 5 with the PGKneo selectable marker cassette. Of 522 ES cell clones screened for targeting by Southern blot analysis, 29 (5%) displayed the predicted restriction fragment length polymorphisms indicative of gene targeting at the E2 locus. As illustrated in Fig. 1A and 1B, an E2 Exon 6 specific probe, which is external to the targeting construct, hybridizes to only a ~16 kb BglI restriction fragment from the wild type allele in the parental R1 ES cell line. In correctly targeted ES cells, this probe also hybridizes to a ~11 kb BglI restriction fragment. Targeting was confirmed with several additional restriction enzymes and probes (data not shown)."}

    craft-ca-core-ex-dev

    {"project":"craft-ca-core-ex-dev","denotations":[{"id":"T10233","span":{"begin":10,"end":12},"obj":"PR_EXT:000006300"},{"id":"T10234","span":{"begin":22,"end":26},"obj":"NCBITaxon:10088"},{"id":"T10235","span":{"begin":36,"end":40},"obj":"SO_EXT:0000704"},{"id":"T10236","span":{"begin":54,"end":59},"obj":"NCBITaxon:10088"},{"id":"T10237","span":{"begin":60,"end":68},"obj":"CL:0002322"},{"id":"T10238","span":{"begin":63,"end":68},"obj":"CL_GO_EXT:cell"},{"id":"T10239","span":{"begin":84,"end":86},"obj":"PR_EXT:000006300"},{"id":"T10240","span":{"begin":87,"end":91},"obj":"SO_EXT:0000704"},{"id":"T10241","span":{"begin":133,"end":135},"obj":"PR_EXT:000006300"},{"id":"T10242","span":{"begin":136,"end":140},"obj":"SO_EXT:0000704"},{"id":"T10243","span":{"begin":172,"end":176},"obj":"SO_EXT:0000704"},{"id":"T10244","span":{"begin":187,"end":196},"obj":"SO_EXT:engineered_biological_sequence"},{"id":"T10245","span":{"begin":213,"end":220},"obj":"SO_EXT:sequence_substitution_process"},{"id":"T10246","span":{"begin":229,"end":230},"obj":"CHEBI_SO_EXT:base"},{"id":"T10247","span":{"begin":242,"end":253},"obj":"SO_EXT:genomic_DNA"},{"id":"T10248","span":{"begin":250,"end":253},"obj":"CHEBI_SO_EXT:DNA"},{"id":"T10249","span":{"begin":276,"end":288},"obj":"SO_EXT:0000852"},{"id":"T10250","span":{"begin":302,"end":306},"obj":"SO_EXT:0000147"},{"id":"T10251","span":{"begin":336,"end":342},"obj":"CHEBI_SO_EXT:molecular_indicator_or_label_or_marker_or_tag"},{"id":"T10252","span":{"begin":360,"end":367},"obj":"CL:0002322"},{"id":"T10253","span":{"begin":363,"end":367},"obj":"CL_GO_EXT:cell"},{"id":"T10254","span":{"begin":368,"end":374},"obj":"SO_EXT:sequence_cloned_entity"},{"id":"T10255","span":{"begin":457,"end":498},"obj":"SO_EXT:0000193"},{"id":"T10256","span":{"begin":513,"end":517},"obj":"SO_EXT:0000704"},{"id":"T10257","span":{"begin":535,"end":537},"obj":"PR_EXT:000006300"},{"id":"T10258","span":{"begin":582,"end":584},"obj":"PR_EXT:000006300"},{"id":"T10259","span":{"begin":585,"end":589},"obj":"SO_EXT:0000147"},{"id":"T10260","span":{"begin":601,"end":606},"obj":"CHEBI_SO_EXT:molecular_probe"},{"id":"T10261","span":{"begin":643,"end":652},"obj":"SO_EXT:engineered_biological_sequence"},{"id":"T10262","span":{"begin":654,"end":664},"obj":"GO:0097617"},{"id":"T10263","span":{"begin":680,"end":681},"obj":"CHEBI_SO_EXT:base"},{"id":"T10264","span":{"begin":687,"end":707},"obj":"SO_EXT:0000412"},{"id":"T10265","span":{"begin":717,"end":726},"obj":"SO_EXT:wild_type_entity_or_quality"},{"id":"T10266","span":{"begin":727,"end":733},"obj":"SO_EXT:0001023"},{"id":"T10267","span":{"begin":753,"end":760},"obj":"CL:0002322"},{"id":"T10268","span":{"begin":756,"end":760},"obj":"CL_GO_EXT:cell"},{"id":"T10269","span":{"begin":789,"end":797},"obj":"CL:0002322"},{"id":"T10270","span":{"begin":792,"end":797},"obj":"CL_GO_EXT:cell"},{"id":"T10271","span":{"begin":804,"end":809},"obj":"CHEBI_SO_EXT:molecular_probe"},{"id":"T10272","span":{"begin":815,"end":825},"obj":"GO:0097617"},{"id":"T10273","span":{"begin":836,"end":837},"obj":"CHEBI_SO_EXT:base"},{"id":"T10274","span":{"begin":843,"end":863},"obj":"SO_EXT:0000412"},{"id":"T10275","span":{"begin":913,"end":932},"obj":"GO_EXT:0015666"},{"id":"T10276","span":{"begin":925,"end":932},"obj":"CHEBI_GO_SO_EXT:enzyme"},{"id":"T10277","span":{"begin":937,"end":943},"obj":"CHEBI_SO_EXT:molecular_probe"}],"text":"To create E2 knockout mice, we used gene targeting in mouse ES cells to disrupt the E2 gene. The overall strategy for disrupting the E2 gene is illustrated in Fig. 1A. The gene targeting construct was designed to replace a 1.67 kb EcoRV/Smal genomic DNA fragment encompassing part of Exon 4 and all of Exon 5 with the PGKneo selectable marker cassette. Of 522 ES cell clones screened for targeting by Southern blot analysis, 29 (5%) displayed the predicted restriction fragment length polymorphisms indicative of gene targeting at the E2 locus. As illustrated in Fig. 1A and 1B, an E2 Exon 6 specific probe, which is external to the targeting construct, hybridizes to only a ~16 kb BglI restriction fragment from the wild type allele in the parental R1 ES cell line. In correctly targeted ES cells, this probe also hybridizes to a ~11 kb BglI restriction fragment. Targeting was confirmed with several additional restriction enzymes and probes (data not shown)."}

    craft-ca-core-dev

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