{"id":668,"date":"2017-03-14T11:53:36","date_gmt":"2017-03-14T02:53:36","guid":{"rendered":"http:\/\/www2.med.osaka-u.ac.jp\/wwwmed\/eng\/activities\/results\/2011year\/horie-kyoji-takeda-junji"},"modified":"2017-04-28T16:07:41","modified_gmt":"2017-04-28T07:07:41","slug":"article01","status":"publish","type":"page","link":"https:\/\/www.med.osaka-u.ac.jp\/eng\/activities\/results\/2011year\/article01","title":{"rendered":"HORIE Kyoji,  TAKEDA Junji \u226aEnvironmental and Molecular Medicine\u226b <span>\u201cA homozygous mutant embryonic stem cell bank applicable for phenotype-driven genetic screening\u201d<\/span>"},"content":{"rendered":"<ul class=\"linkBar clearfix\">\n<li> <a href=\"http:\/\/www.med.osaka-u.ac.jp\/activities\/results\/2011year\/article01\">Text in Japanese<\/a><\/li>\n<\/ul>\n<p><!-- \n\n<h1> \u5800\u6c5f \u606d\u4e8c\u3001\u7af9\u7530 \u6f64\u4e8c\u226a\u74b0\u5883\u30fb\u751f\u4f53\u6a5f\u80fd\u5b66\u226b<\/h1>\n\n --><\/p>\n<p class=\"figure\"><a href=\"http:\/\/www.med.osaka-u.ac.jp\/eng\/wp-content\/uploads\/\/activities\/results\/2011\/horie20111023_s.jpeg\" target=\"_blank\" class=\"zoom\"><img loading=\"lazy\" decoding=\"async\" src=\"http:\/\/www.med.osaka-u.ac.jp\/eng\/wp-content\/uploads\/\/activities\/results\/2011\/horie20111023_s.jpeg\" alt=\"\u74b0\u5883\u30fb\u751f\u4f53\u6a5f\u80fd\u5b66\" height=\"237\" width=\"234\" \/><br \/>\n  <span class=\"click\">Click to enlarge<\/span><\/a><\/p>\n<p>2011-10-23<br \/>\n  <span class=\"lineFrame\">Publish<\/span>Nature Methods. 8: 1071-1077 (2011)<\/p>\n<p><!-- \n\n<h3>\u8868\u73fe\u578b\u306e\u8fc5\u901f\u306a\u30b9\u30af\u30ea\u30fc\u30cb\u30f3\u30b0\u304c\u53ef\u80fd\u306a\u30db\u30e2\u5909\u7570\u4f53ES\u7d30\u80de\u30d0\u30f3\u30af\u306e\u4f5c\u88fd<\/h3>\n\n\n\n\n<p> \u30de\u30a6\u30b9ES\u7d30\u80de\u306b\u304a\u3051\u308b\u30b2\u30ce\u30e0\u30ef\u30a4\u30c9\u306a\u5909\u7570\u5c0e\u5165\u6cd5\u306f\u3001\u907a\u4f1d\u5b50\u6a5f\u80fd\u89e3\u6790\u3092\u884c\u3046\u4e0a\u3067\u3001\u6709\u52b9\u306a\u624b\u6cd5\u3067\u3042\u308b\u3002\u3057\u304b\u3057\u3001\u54fa\u4e73\u52d5\u7269\u7d30\u80de\u306e\u30b2\u30ce\u30e0\u306b\u306f\u3001\u907a\u4f1d\u5b50\u304c\uff12\u30b3\u30d4\u30fc\u305a\u3064\u5b58\u5728\u3059\u308b\u305f\u3081\u3001\u8868\u73fe\u578b\u3092\u9732\u5448\u3055\u305b\u308b\u306b\u306f\u4e21\u65b9\u306e\u30b3\u30d4\u30fc\u3092\u7834\u58ca\u3059\u308b\u5fc5\u8981\u304c\u3042\u308a\u3001\u89e3\u6790\u306e\u8fc5\u901f\u5316\u306e\u59a8\u3052\u3068\u306a\u3063\u3066\u3044\u308b\u3002\u3053\u306e\u554f\u984c\u3092\u89e3\u6c7a\u3059\u308b\u305f\u3081\u306b\u3001\u6211\u3005\u306f\u4ee5\u524d\u306b\u3001Bloom\u75c7\u5019\u7fa4\u539f\u56e0\u907a\u4f1d\u5b50\u306e\u767a\u73fe\u3092\u4e00\u904e\u6027\u306b\u4f4e\u4e0b\u3055\u305b\u3066\u3001\u30d8\u30c6\u30ed\u5909\u7570\u4f53ES\u7d30\u80de\u304b\u3089\u30db\u30e2\u5909\u7570\u4f53ES\u7d30\u80de\u3092\u8a98\u767a\u3059\u308b\u65b9\u6cd5\u3092\u5831\u544a\u3057\u305f\u3002\u3057\u304b\u3057\u3001\u8a98\u767a\u3057\u305f\u30db\u30e2\u5909\u7570\u4f53\u3092\u30d8\u30c6\u30ed\u5909\u7570\u4f53\u304b\u3089\u5358\u96e2\u3067\u304d\u306a\u304b\u3063\u305f\u305f\u3081\u3001\u30db\u30e2\u5909\u7570\u4f53\u3068\u30d8\u30c6\u30ed\u5909\u7570\u4f53\u304c\u6df7\u5728\u3059\u308b\u72b6\u614b\u3067\u8868\u73fe\u578b\u89e3\u6790\u3092\u884c\u3046\u5fc5\u8981\u304c\u3042\u308a\u3001\u9069\u7528\u3067\u304d\u308b\u8868\u73fe\u578b\u304c\u9650\u3089\u308c\u3066\u3044\u305f\u3002\u672c\u8ad6\u6587\u3067\u306f\u3001\u30db\u30e2\u5909\u7570\u4f53ES\u7d30\u80de\u3092\u52b9\u7387\u7684\u306b\u5358\u96e2\u3059\u308b\u65b9\u6cd5\u3068\u3001\u8868\u73fe\u578b\u89e3\u6790\u3078\u306e\u6709\u52b9\u6027\u3092\u5831\u544a\u3059\u308b\u3002\u307e\u305a\u3001\u30db\u30e2\u5909\u7570\u4f53\u3092G418\/puromycin\u8010\u6027\u682a\u3068\u3057\u3066\u9078\u629e\u3059\u308b\u305f\u3081\u306e\u3001\u65b0\u305f\u306a\u85ac\u5264\u8010\u6027\u907a\u4f1d\u5b50\u30ab\u30bb\u30c3\u30c8\u3092\u958b\u767a\u3057\u305f\u3002\u3055\u3089\u306b\u3001\u5909\u7570\u90e8\u4f4d\u306b\u9023\u9396\u3057\u305fSNP\u3092\u89e3\u6790\u3059\u308b\u3053\u3068\u3067\u3001\u30db\u30e2\u5909\u7570\u4f53\u306e\u9078\u629e\u52b9\u7387\u3092\u9ad8\u3081\u305f\u3002\u3053\u306e\u624b\u6cd5\u306b\u3001\u30b8\u30fc\u30f3\u30c8\u30e9\u30c3\u30d7\u30d9\u30af\u30bf\u30fc\u306b\u3088\u308brandom mutagenesis\u3092\u9069\u7528\u3057\u3001\u30db\u30e2\u5909\u7570\u4f53ES\u7d30\u80de\u30d0\u30f3\u30af\u3092\u69cb\u7bc9\u3057\u305f\u3002\u3053\u306e\u30d0\u30f3\u30af\u3092\u5229\u7528\u3057\u3066\u3001\u591a\u5206\u5316\u80fd\u3001\u5897\u6b96\u6027\u306a\u3069\u306b\u7570\u5e38\u3092\u793a\u3059\u69d8\u3005\u306a\u5909\u7570\u4f53\u3092\u540c\u5b9a\u3057\u3001\u7d30\u80de\u30ec\u30d9\u30eb\u3067\u306e\u8868\u73fe\u578b\u89e3\u6790\u3092\u8fc5\u901f\u306b\u884c\u3048\u308b\u3053\u3068\u3092\u793a\u3057\u305f\u3002<\/p>\n\n --><\/p>\n<p> Genome-wide mutagenesis in mouse embryonic stem cells (ESCs) is a powerful tool, but the diploid nature of the mammalian genome hampers its application for recessive genetic screening. We have previously reported a method to induce homozygous mutant ESCs from heterozygous mutants by tetracycline-dependent transient disruption of the Bloom&#8217;s syndrome gene. However, we could not purify homozygous mutants from a large population of heterozygous mutant cells, limiting the applications. Here we developed a strategy for rapid enrichment of homozygous mutant mouse ESCs and demonstrated its feasibility for cell-based phenotypic analysis. The method uses G418-plus-puromycin double selection to enrich for homozygotes and single-nucleotide polymorphism analysis for identification of homozygosity. We combined this simple approach with gene-trap mutagenesis to construct a homozygous mutant ESC bank and demonstrate its use in phenotype-driven genetic screening.<\/p>\n<p><span class=\"lineFrame\">URL<\/span><a href=\"http:\/\/www.med.osaka-u.ac.jp\/pub\/mr-envi\/www\/\" target=\"_blank\">http:\/\/www.med.osaka-u.ac.jp\/pub\/mr-envi\/www\/<\/a><\/p>\n","protected":false},"excerpt":{"rendered":"<p>Text in Japanese Click to enlarge 2011-10-23 PublishNature Methods. 8: 1071-1077 (2011) Genome-wide mutagenesi [&hellip;]<\/p>\n","protected":false},"author":1,"featured_media":1791,"parent":460,"menu_order":136,"comment_status":"closed","ping_status":"closed","template":"","meta":{"footnotes":""},"_links":{"self":[{"href":"https:\/\/www.med.osaka-u.ac.jp\/eng\/wp-json\/wp\/v2\/pages\/668"}],"collection":[{"href":"https:\/\/www.med.osaka-u.ac.jp\/eng\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/www.med.osaka-u.ac.jp\/eng\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/www.med.osaka-u.ac.jp\/eng\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/www.med.osaka-u.ac.jp\/eng\/wp-json\/wp\/v2\/comments?post=668"}],"version-history":[{"count":6,"href":"https:\/\/www.med.osaka-u.ac.jp\/eng\/wp-json\/wp\/v2\/pages\/668\/revisions"}],"predecessor-version":[{"id":2386,"href":"https:\/\/www.med.osaka-u.ac.jp\/eng\/wp-json\/wp\/v2\/pages\/668\/revisions\/2386"}],"up":[{"embeddable":true,"href":"https:\/\/www.med.osaka-u.ac.jp\/eng\/wp-json\/wp\/v2\/pages\/460"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/www.med.osaka-u.ac.jp\/eng\/wp-json\/wp\/v2\/media\/1791"}],"wp:attachment":[{"href":"https:\/\/www.med.osaka-u.ac.jp\/eng\/wp-json\/wp\/v2\/media?parent=668"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}