{"id":157,"date":"2023-02-26T02:00:47","date_gmt":"2023-02-25T18:00:47","guid":{"rendered":"http:\/\/39.105.184.3\/?p=157"},"modified":"2025-12-21T01:06:34","modified_gmt":"2025-12-20T17:06:34","slug":"%e4%bd%bf%e7%94%a8knockout-first%e6%89%93%e9%9d%b6%e7%ad%96%e7%95%a5%e6%9e%84%e5%bb%ba%e5%9f%ba%e5%9b%a0%e6%95%b2%e9%99%a4%e9%bc%a0","status":"publish","type":"post","link":"https:\/\/fanyiming.life\/?p=157","title":{"rendered":"\u4f7f\u7528Knockout-First\u6253\u9776\u7b56\u7565\u6784\u5efa\u57fa\u56e0\u6572\u9664\u9f20"},"content":{"rendered":"\n<blockquote class=\"wp-block-quote is-layout-flow wp-block-quote-is-layout-flow\">\n<p>\u672c\u6587\u662f\u7f51\u4e0a\u5404\u79cd\u8d44\u6599\u4e1c\u6284\u6284\u897f\u6284\u6284\u6574\u7406\u7684\uff0c\u4e3a\u4e86\u7701\u4e8b\u5c31\u4e0d\u4e00\u4e00\u5bf9\u5e94\u5730\u5217\u51fa\u5904\u4e86\uff0c\u53c2\u8003\u94fe\u63a5\u7edf\u4e00\u5728\u6587\u672b\u3002<\/p>\n<\/blockquote>\n\n\n\n<h1 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class=\"wp-block-list\">\n<li>loxP: \u5168\u79f0locus of X-over P1\uff0c\u6765\u6e90\u4e8e\u566c\u83cc\u4f53P1\u3002\u662f\u4e00\u6bb5\u957f\u5ea6\u4e3a34bp\u7684DNA\u5e8f\u5217\uff0c\u5305\u62ec\u4e24\u4e2a13bp\u7684\u53cd\u5411\u91cd\u590d\u5e8f\u5217\u548c\u4e00\u4e2a\u4e0d\u5bf9\u79f0\u76848bp\u95f4\u9694\u533a\u7ec4\u6210\u3002\u53cd\u5411\u91cd\u590d\u5e8f\u5217\u662fCre\u91cd\u7ec4\u9176\u7684\u7279\u5f02\u8bc6\u522b\u4f4d\u70b9\uff0c\u800c\u95f4\u9694\u533a\u57df\u51b3\u5b9a\u4e86loxP\u4f4d\u70b9\u7684\u65b9\u5411\u3002\uff08ATAACTTCGTATA &#8211; NNNTANNN -TATACGAAGTTAT\uff09<\/li>\n\n\n\n<li>Cre: 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src=\"https:\/\/eamonimgbed.oss-cn-beijing.aliyuncs.com\/img\/202302261506258.webp?x-oss-process=style\/screenshots\" alt=\"img\" \/><\/figure>\n\n\n\n<p><\/p>\n\n\n\n<p>\u5728\u6784\u5efa\u6761\u4ef6\u6027\u57fa\u56e0\u6572\u9664\u9f20\u65f6\uff0c\u901a\u5e38\u5c06\u7f16\u7801Cre\u9176\u7684\u57fa\u56e0\u548c\u5e26\u6709loxP\u4f4d\u70b9\u7684\u57fa\u56e0\u653e\u5728\u4e0d\u540c\u7684\u8001\u9f20\u4e0a\uff08\u4e8c\u8005\u5206\u522b\u79f0\u4e3aCre\u5c0f\u9f20\u548cflox\u5c0f\u9f20\uff09\uff0c\u7136\u540e\u901a\u8fc7\u6742\u4ea4\u7684\u65b9\u6cd5\u8ba9\u5b83\u4eec\u5728\u5b50\u4ee3\u4e2d\u76f8\u9047\uff0c\u5c06Cre\u9176\u7684coding 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Blotting\u7b49\u65b9\u6cd5\u9274\u5b9a\u662f\u5426\u6b63\u786e\u53d1\u751f\u540c\u6e90\u91cd\u7ec4<\/li>\n\n\n\n<li>\u5c06\u4e2d\u9776ES\u7ec6\u80de\u6ce8\u5c04\u5230\u53d7\u4f53\u56ca\u80da\u4e2d\uff0c\u5c06\u6ce8\u5c04\u540e\u5b58\u6d3b\u7684\u80da\u80ce\u79fb\u690d\u5230\u5047\u5b55\u5c0f\u9f20\u5b50\u5bab\u4e2d\uff0c\u8ba9\u5176\u53d7\u5b55\u3002<\/li>\n\n\n\n<li>\u5c0f\u9f20\u51fa\u751f\u540e\uff0c\u901a\u8fc7\u89c2\u5bdf\u6bdb\u8272\u662f\u5426\u5d4c\u5408\u548c\u5d4c\u5408\u7a0b\u5ea6\u5224\u65ad\u662f\u5426\u83b7\u5f97\u5d4c\u5408\u4f53\u5c0f\u9f20\u3002<\/li>\n<\/ol>\n\n\n\n<p>\u56ca\u80da\u8154\u4e2d\u6709\u5f88\u591aES\u7ec6\u80de\uff0c\u6211\u4eec\u6ce8\u5c04\u8fdb\u53bb\u7684\u4e5f\u662fES\u7ec6\u80de\uff0c\u4e3a\u4e86\u533a\u5206\uff0c\u901a\u5e38\u4f7f\u7528\u4e0d\u540c\u6bdb\u8272\u7684ES\u7ec6\u80de\u548c\u4f9b\u4f53\u56ca\u80da\u3002\u8fd9\u6837\uff0c\u5982\u679c\u5c0f\u9f20\u6bdb\u8272\u4e2d\u51fa\u73b0\u4e86ES\u7ec6\u80de\u7684\u6bdb\u8272\uff0c\u8bf4\u660e\u5d4c\u5408\u6210\u529f\u3002\u6ce8\u610f\u533a\u5206\u5d4c\u5408\u4f53\u548c\u6742\u5408\u5b50\uff0c\u6742\u5408\u5b50\u662f\u540c\u6e90\u67d3\u8272\u4f53\u4e0a\u7684\u4e24\u4e2a\u7b49\u4f4d\u57fa\u56e0\u4e0d\u540c\uff0c\u5d4c\u5408\u5b50\u662f\u5168\u8eab\u4e0a\u4e0b\u7684\u90a3\u4e48\u591a\u7ec6\u80de\u5206\u522b\u6765\u6e90\u4e8e\u4e0d\u540c\u80cc\u666f\u7684ES\u7ec6\u80de\u3002\u5d4c\u5408\u4f53\u4e0d\u80fd\u76f4\u63a5\u62ff\u6765\u505a\u5b9e\u9a8c\uff0c\u88ab\u79f0\u4e3aF0\u4ee3\u5c0f\u9f20\u3002\u5c06F0\u4e0e\u91ce\u751f\u578b\u5c0f\u9f20\u6742\u4ea4\uff0c\u901a\u8fc7\u5bf9\u5b50\u4ee3PCR\u9274\u5b9aF0\u4e2d\u7684\u4e2d\u9776\u57fa\u56e0\u662f\u5426\u5177\u6709\u751f\u6b96\u7cfb\u9057\u4f20\u80fd\u529b\u3002\u8fd9\u6837\u83b7\u5f97\u7684\u662f\u6742\u5408\u5b50F1\u4ee3\u5c0f\u9f20\u3002\u4e4b\u540e\u6839\u636e\u5b9e\u9a8c\u76ee\u7684\u7ee7\u7eed\u6742\u4ea4\u5f97\u5230\u7eaf\u5408\u6572\u9664\u5c0f\u9f20\u6216\u8fde\u7eed\u56de\u4ea4\uff08\u4e00\u822c\u662f10\u4ee3\uff09\u6765\u7eaf\u5316\u9057\u4f20\u80cc\u666f\u3002<\/p>\n\n\n\n<p><\/p>\n\n\n\n<p>CRISPR\/Cas9\u6253\u9776\u7684\u8fc7\u7a0b\uff1a<\/p>\n\n\n\n<ol class=\"wp-block-list\">\n<li>\u9488\u5bf9\u57fa\u56e0\u7ec4\u7279\u5f02\u5e8f\u5217\u8bbe\u8ba1\u5411\u5bfcRNA (gRNA)<\/li>\n\n\n\n<li>\u4f53\u5916\u8f6c\u5f55gRNA\u4ee5\u53ca\u7f16\u7801Cas9\u86cb\u767d\u7684mRNA<\/li>\n\n\n\n<li>\u5c06gRNA, Cas9 mRNA\u6ce8\u5c04\u5230\u5c0f\u9f20\u53d7\u7cbe\u5375\u4e2d<\/li>\n\n\n\n<li>\u6ce8\u5c04\u540e\u7684\u53d7\u7cbe\u5375\u79fb\u690d\u5230\u5047\u5b55\u6bcd\u9f20\u8f93\u5375\u7ba1\u4e2d\uff0c\u53d7\u7cbe\u5375\u53d1\u80b2\u6210F0\u4ee3\u5c0f\u9f20<\/li>\n<\/ol>\n\n\n\n<p>\u5982\u679c\u4e0d\u5728\u53d7\u7cbe\u5375\u4e2d\u989d\u5916\u6ce8\u5c04\u542b\u6709\u540c\u6e90\u81c2\u7684\u57fa\u56e0\u66ff\u6362\u76d2\uff0c\u53ea\u662f\u8ba9DNA\u5728\u88abCas9\u5207\u5272\u540e\u4f9d\u9760\u975e\u540c\u6e90\u672b\u7aef\u4fee\u590d\uff0c\u8fd9\u6837\u5c31\u662f\u5168\u8eab\u6572\u9664\u3002\u867d\u7136\u662f\u5728\u53d7\u7cbe\u5375\uff081\u7ec6\u80de\u671f\uff09\u6ce8\u5c04\u7684\uff0c\u4f46\u662f\u8fd9\u4e2a\u65b9\u6cd5\u4ecd\u7136\u53ef\u80fd\u5f97\u5230\u5d4c\u5408\u4f53\u3002\u56e0\u4e3aCas9\u53d1\u6325\u4f5c\u7528\u6709\u65f6\u95f4\u4e0a\u7684\u5ef6\u540e\uff0c\u53ef\u80fd\u57282\u7ec6\u80de\u671f\u62164\u7ec6\u80de\u671f\u624d\u5f00\u59cb\u5207\u5272DNA\uff0c\u6216\u8005\u867d\u7136\u8fd8\u672a\u52302\u7ec6\u80de\u671f\uff0c\u4f46DNA\u590d\u5236\u5df2\u7ecf\u5f00\u59cb\uff0c\u5c31\u53ef\u80fd\u9020\u6210\u9776\u57fa\u56e0\u7684\u7a81\u53d8\u7c7b\u578b\u5728\u4e0d\u540c\u540e\u4ee3\u7ec6\u80de\u4e2d\u4e0d\u540c\u3002<\/p>\n\n\n\n<p>\u5982\u679c\u5728\u53d7\u7cbe\u5375\u4e2d\u989d\u5916\u6ce8\u5c04\u542b\u6709\u540c\u6e90\u81c2\u7684\u57fa\u56e0\u66ff\u6362\u76d2\uff0c\u90a3\u5c31\u53ef\u4ee5\u901a\u8fc7\u540c\u6e90\u672b\u7aef\u4fee\u590d\u5f15\u5165\u4e24\u7ffc\u6709loxP site\u7684DNA\u7247\u6bb5\uff0c\u6784\u5efaflox\u5c0f\u9f20\uff0c\u4ece\u800c\u7528\u4e8e\u6761\u4ef6\u6027\u57fa\u56e0\u6572\u9664\u3002<\/p>\n\n\n\n<p><\/p>\n\n\n\n<p>ES\u6253\u9776\u548cCRISPR\/Cas9\u6253\u9776\u7684\u5bf9\u6bd4\uff1a<\/p>\n\n\n\n<figure class=\"wp-block-image\"><img loading=\"lazy\" decoding=\"async\" decoding=\"async\" src=\"https:\/\/eamonimgbed.oss-cn-beijing.aliyuncs.com\/img\/202302261507038.webp?x-oss-process=style\/screenshots\" alt=\"img\" \/><\/figure>\n\n\n\n<p>\uff08\u7b2c\u4e09\u5217\u90a3\u4e2aTurboKnockout\u662f\u8d5b\u4e1a\u751f\u7269\u5f00\u53d1\u7684\u53e6\u4e00\u4e2a\u65b9\u6cd5\uff0c\u672c\u6587\u5148\u4e0d\u63d0\u3002\u4f9d\u9760\u975e\u540c\u6e90\u672b\u7aef\u4fee\u590d\u65b9\u5f0f\u7684\u8f6c\u57fa\u56e0(Transgenic)\u5c0f\u9f20\u56e0\u4e3a\u57fa\u56e0\u7684\u63d2\u5165\u65b9\u5f0f\u6216\u7a81\u53d8\u7c7b\u578b\u5e76\u4e0d\u5355\u4e00\uff0c\u6240\u4ee5\u53ef\u80fd\u8868\u578b\u4e5f\u4f1a\u6709\u5dee\u5f02\u3002\u800c\u4f9d\u9760\u540c\u6e90\u91cd\u7ec4\u65b9\u5f0f\u7684Knock-in\u6216Knock-out\u5c0f\u9f20\u7684Genotype\u662f\u5b8c\u5168\u4e00\u81f4\u7684\u3002\uff09<\/p>\n\n\n\n<p>Cre-loxP\u5e76\u4e0d\u662f\u552f\u4e00\u7684\u91cd\u7ec4\u9176\u7cfb\u7edf\uff0cFLP-FRT\u7cfb\u7edf\u662f\u4e0eCre-loxP\u7c7b\u4f3c\u7684\u91cd\u7ec4\u9176\u7cfb\u7edf\u3002Cre-loxP\u4ece\u566c\u83cc\u4f53\u4e2d\u53d1\u73b0\uff0cFLP-FRT\u4ece\u917f\u9152\u9175\u6bcd\u4e2d\u53d1\u73b0\u3002<\/p>\n\n\n\n<ul class=\"wp-block-list\">\n<li>FLP: flippase(FLP) recombinase, \u8bc6\u522b\u4e00\u5bf9FRT\u4f4d\u70b9\u4e4b\u95f4\u7684DNA\u5e8f\u5217<\/li>\n\n\n\n<li>FRT: FLP recombinase target\uff0c\u7c7b\u6bd4loxP site<\/li>\n<\/ul>\n\n\n\n<h1 class=\"wp-block-heading\">Knockout-First\u7b56\u7565<\/h1>\n\n\n\n<p>Knockout-First\u7b56\u7565\u662f\u4e00\u79cd\u7ed3\u5408\u4e86Cre-loxP\u548cFLP-FRT\u7684\u5c0f\u9f20\u57fa\u56e0\u6572\u9664\u7b56\u7565\u3002\u5b83\u7684\u57fa\u56e0\u66ff\u6362\u76d2\u7684\u6838\u5fc3\u533a\u57df\u957f\u4e0b\u9762\u8fd9\u6837\uff1a<\/p>\n\n\n\n<figure class=\"wp-block-image\"><img loading=\"lazy\" decoding=\"async\" decoding=\"async\" src=\"https:\/\/eamonimgbed.oss-cn-beijing.aliyuncs.com\/img\/202302261507147.png?x-oss-process=style\/screenshots\" alt=\"image-20230226150745139\" \/><\/figure>\n\n\n\n<p>\u200bsA, splice acceptor element; IRES, internal ribosomal entry site; lacZ, gene encoding \u03b2-galactosidase; neo, neomycin phosphotransferase; pA, SV40 polyadenylation signal<\/p>\n\n\n\n<p>\u200b<\/p>\n\n\n\n<p>\u628a\u8fd9\u4e2a\u5e8f\u5217\u63d2\u5165\u57fa\u56e0\u5185\u90e8\u7684\u5185\u542b\u5b50\uff0csplice acceptor element\u5c31\u4f1a\u5728\u526a\u63a5\u65f6\u622a\u83b7\u8fd9\u6761mRNA\uff0c\u4f7f\u5b83\u62fc\u63a5\u5230\u8fd9\u6bb5\u5e8f\u5217\u4e0a\u3002IRES\u662f\u5185\u90e8\u6838\u7cd6\u4f53\u8fdb\u5165\u4f4d\u70b9\uff0c\u4f7fLacZ\u548cNeo\u53ef\u4ee5\u6b63\u5e38\u7ffb\u8bd1\u3002\u4e4b\u540e\u7684SV40 polyA signal\u4f7f\u8f6c\u5f55\u5230\u8fd9\u91cc\u65f6\u7ec8\u6b62\uff0c\u4e8e\u662f\u63d2\u5165\u5e8f\u5217\u540e\u9762\u7684\u540e\u534a\u6bb5\u57fa\u56e0\u5c31\u4e0d\u4f1a\u8868\u8fbe\u4e86\u3002\u56e0\u4e3a\u4f9d\u8d56\u7684\u662f\u4eba\u4e3a\u6539\u53d8\u526a\u63a5\u65b9\u5f0f\uff0c\u6574\u6bb5\u57fa\u56e0\u7684\u8868\u8fbe\u4f9d\u7136\u662f\u5728\u9776\u57fa\u56e0\u542f\u52a8\u5b50\u7684\u63a7\u5236\u4e0b\u7684\uff0c\u5728Neo\u524d\u63d2\u5165Tn903 promoter\uff0c\u53ef\u80fd\u662f\u4e3a\u4e86\u5e94\u5bf9\u9776\u57fa\u56e0\u672c\u8eab\u8868\u8fbe\u91cf\u4f4e\u65f6\uff0cNeo\u4ecd\u53ef\u4ee5\u4f5c\u4e3a\u7b5b\u9009\u6807\u8bb0\u88ab\u6b63\u5e38\u8868\u8fbe\u3002<\/p>\n\n\n\n<p>\u53ef\u4ee5\u770b\u5230\uff0c\u53ea\u8981\u8fd9\u6bb5\u5e8f\u5217\u88ab\u63d2\u5165\u5230\u9776\u57fa\u56e0\u5185\u90e8\uff0c\u5373\u4fbf\u9776\u57fa\u56e0\u7684\u6240\u6709\u5916\u663e\u5b50\u4ecd\u7136\u662f\u5b8c\u6574\u7684\uff0c\u5b83\u7684\u8868\u8fbe\u4ecd\u7136\u88ab\u7834\u574f\u4e86\u3002\u56e0\u6b64\uff0cKnock-First\u53ef\u4ee5\u5b9e\u73b0\u5b8c\u5168\u6027\u57fa\u56e0\u6572\u9664\u3002\u800c\u5728\u4e0a\u8ff0\u6838\u5fc3\u5143\u4ef6\u7684\u57fa\u7840\u4e0a\uff0c\u501f\u52a9Cre-loxP\u548cFLP-FRT\uff0c\u5b83\u540c\u65f6\u4e5f\u53ef\u4ee5\u8f6c\u6362\u4e3a\u6761\u4ef6\u6027\u57fa\u56e0\u6572\u9664\u3002<\/p>\n\n\n\n<figure class=\"wp-block-image\"><img loading=\"lazy\" decoding=\"async\" decoding=\"async\" src=\"https:\/\/eamonimgbed.oss-cn-beijing.aliyuncs.com\/img\/202302261508528.png?x-oss-process=style\/screenshots\" alt=\"image-20230226150828158\" \/><\/figure>\n\n\n\n<p>\u5982\u56fe\uff0cTargeting vector\u662f\u9700\u8981\u6784\u5efa\u7684\u57fa\u56e0\u66ff\u6362\u76d2\uff0c\u901a\u8fc7\u4e24\u6bb5\u201chomology arm\u201d\u548cWildtype allele\u8fdb\u884c\u540c\u6e90\u91cd\u7ec4\uff0c\u5c31\u5f97\u5230KO-First allele\uff08tm1a\uff09\u3002\u6b64\u65f6\uff0c\u7531\u4e8e\u7eff\u8272\u65b9\u6846\u8868\u793a\u7684SA-IRES-Reporter-polyA\uff0c\u8fd9\u6761allele\u4e0a\u7684\u9776\u57fa\u56e0\u5df2\u7ecf\u88ab\u6572\u9664\uff0c\u6240\u4ee5\u8fd9\u4e2a\u6280\u672f\u88ab\u53eb\u505a\u201cKnockout-First\u201d\uff08\u76f8\u5bf9\u4e8e\u4f20\u7edf\u6784\u5efaFlox\u5c0f\u9f20\u7684\u201cConditional-First\u201d)\u3002\u800c\u5728\u6838\u5fc3\u5143\u4ef6\u5185\u90e8\u548c\u5468\u56f4\u6309\u7167\u56fe\u793a\uff0c\u63d2\u5165\u4e862\u4e2aFRT\u4f4d\u70b9\u548c3\u4e2aloxP\u4f4d\u70b9\u3002<\/p>\n\n\n\n<p>\u6b64\u65f6\u6709\u4e24\u79cd\u60c5\u51b5\uff0c\u5982\u679c\u5148\u548cFlp\u5c0f\u9f20\u6742\u4ea4\uff0c2\u4e2aFRT\u4e4b\u95f4\u7684\u90e8\u5206\u88ab\u5220\u9664\uff0c\u5b8c\u5168\u6027\u6572\u9664\u7684tm1a\u53d8\u6210\u6761\u4ef6\u6027\u6572\u9664\u7684tm1c\uff0c\u5728\u6ca1\u6709Cre\u9176\u65f6\uff0ctm1c\u4e0a\u7684\u9776\u57fa\u56e0\u662f\u6b63\u5e38\u8868\u8fbe\u7684\uff0c\u5f53\u9047\u5230Cre\u65f6\uff0ctm1c\u4e0a\u76842\u4e2aloxP\u4f4d\u70b9\u4e4b\u95f4\u7684\u5e8f\u5217\u88ab\u5220\u9664\uff0c\u6210\u4e3a\u9776\u57fa\u56e0\u6572\u9664\u7684tm1d\u3002\u800c\u5982\u679ctm1a\u5148\u56e0\u4e0eCre\u5c0f\u9f20\u6742\u4ea4\uff0c3\u4e2aloxP\u4f4d\u70b9\u4e4b\u95f4\u7684\u5e8f\u5217\u76f4\u63a5\u88ab\u5220\u9664\uff0c\u5c31\u53d8\u6210tm1b\uff0ctm1b\u4e0a\u7684\u9776\u57fa\u56e0\u4e5f\u76f4\u63a5\u88ab\u6572\u9664\u3002<\/p>\n\n\n\n<p><strong>KO-First Alleles: Generic Mutation Information<\/strong><\/p>\n\n\n\n<figure class=\"wp-block-table\"><table><thead><tr><th>** Mutation**<\/th><th><strong>Allele<\/strong><\/th><th><strong>Description<\/strong><\/th><th><strong>Conditional Potential<\/strong><\/th><\/tr><\/thead><tbody><tr><td>tm1a<\/td><td>Knockout-First(KO-First)<\/td><td>Reporter-tagged insertion allele (Null)<\/td><td>Yes<\/td><\/tr><tr><td>tm1b<\/td><td>CREed KO-First<\/td><td>Reporter-tagged deletion allele (for IMPC)<\/td><td>No<\/td><\/tr><tr><td>tm1c<\/td><td>FLPed KO-First<\/td><td>Conditional ready (restored gene activity)<\/td><td>Yes<\/td><\/tr><tr><td>tm1d<\/td><td>FLPed, CREed KO-First<\/td><td>Deletion allele, no reporter (Null)<\/td><td>No<\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\n<p>\u6839\u636e\u5b9e\u9a8c\u76ee\u7684\u7684\u9700\u8981\uff0c\u53ef\u4ee5\u5bf9KO-First\u5c0f\u9f20\u8fdb\u884c\u4e0d\u540c\u7684\u6742\u4ea4\u7b56\u7565\u6765\u8fbe\u5230\u4e0d\u540c\u76ee\u7684\uff0c\u65e2\u53ef\u4ee5\u5b8c\u5168\u6027\u6572\u9664\uff0c\u4e5f\u53ef\u4ee5\u901a\u8fc7FLP\u91cd\u7ec4\u8f6c\u53d8\u4e3a\u6761\u4ef6\u6027\u6572\u9664\uff0c\u5b9e\u73b0\u4e00\u9f20\u591a\u7528\u3002<\/p>\n\n\n\n<p>\u4e0b\u9762\u4e3e\u4e00\u4e2a\u5229\u7528knockout-first\u7684\u5177\u4f53\u4f8b\u5b50\uff0c\u5047\u8bbe\u6211\u4eec\u9700\u8981\u5229\u7528KO-First\u7b56\u7565\u6784\u5efa\u57fa\u56e0X\u7684\u6761\u4ef6\u6027\u57fa\u56e0\u6572\u9664\u5c0f\u9f20\uff0c\u9700\u8981\u5982\u4f55\u6784\u5efaKO-First\u5c0f\u9f20\u548c\u8bbe\u8ba1\u540e\u7eed\u7684\u6742\u4ea4\u5b9e\u9a8c\u5462\uff1f<\/p>\n\n\n\n<ol class=\"wp-block-list\">\n<li>\u5df2\u77e5\u6572\u9664\u57fa\u56e0X\u7684Exon2\u53ef\u4ee5\u4f7f\u5176\u529f\u80fd\u5931\u6d3b\uff0c\u6240\u4ee5\u6211\u4eec\u636e\u6b64\u6784\u5efa\u597d\u4e86\u5305\u56f4Exon2\u7684KO-First Targeting Vector\u3002<img loading=\"lazy\" decoding=\"async\" decoding=\"async\" alt=\"image-20230226150900423\" src=\"https:\/\/eamonimgbed.oss-cn-beijing.aliyuncs.com\/img\/202302261509384.png?x-oss-process=style\/screenshots\"> \uff08promoter\u548cpromoterless\u7684\u4e0d\u540c\u7528\u9014\u6211\u8fd8\u6ca1\u641e\u6e05\u695a)<\/li>\n\n\n\n<li>\u9009\u62e9\u6765\u6e90\u4e8eC57BL\/6N\u5c0f\u9f20\u7684ES\u7ec6\u80de\u7cfbJM8.N4\uff0c\u6bdb\u8272\u662f\u9ed1\u8272\u3002\u5c06Targeting Vector\u663e\u5fae\u6ce8\u5c04\u5230\u82e5\u5e72ES\u7ec6\u80de\u4e2d\uff0c\u52a0\u836f\u7b5b\u9009\u3002\u7b5b\u5230\u7684ES\u7ec6\u80de\u6269\u589e\u57f9\u517b\uff0c\u5e76\u7528PCR\u9274\u5b9a\u662f\u5426\u6210\u529f\u4e2d\u9776\u3002<\/li>\n\n\n\n<li>\u5c06\u6210\u529f\u4e2d\u9776\u7684ES\u7ec6\u80de\u6ce8\u5c04\u5230\u4f9b\u4f53\u56ca\u80da\u4e2d\uff08\u6bdb\u8272\u68d5\u8272\uff09\uff0c\u5c06\u5904\u7406\u540e\u7684\u56ca\u80da\u624b\u672f\u653e\u5165\u5047\u5b55\u96cc\u9f20\u7684\u5b50\u5bab\u3002\u8ba9\u5b83\u751f\uff0c\u5f97\u5230\u5d4c\u5408\u4f53\u5c0f\u9f20\uff08F0\uff09\u3002<\/li>\n\n\n\n<li>\u901a\u8fc7\u6bdb\u8272\u5d4c\u5408\u60c5\u51b5\uff0c\u9009\u62e9\u9ed1\u8272\u5d4c\u5408\u5ea6\u9ad8\u7684\u5c0f\u9f20\uff0c\u4e0e\u91ce\u751f\u578b\u7684\u80cc\u666f\u5c0f\u9f20C57BL\/6N\u6742\u4ea4\u3002<img loading=\"lazy\" decoding=\"async\" decoding=\"async\" alt=\"image-20230226152626228\" src=\"https:\/\/eamonimgbed.oss-cn-beijing.aliyuncs.com\/img\/202302261526880.png?x-oss-process=style\/screenshots\">\u7406\u60f3\u60c5\u51b5\u4e0b\uff0cF1\u4f1a\u662f\u4e0b\u56fe\u6240\u793a\u7684\u6742\u5408\u5b50\u3002<img loading=\"lazy\" decoding=\"async\" decoding=\"async\" alt=\"image-20230226152712313\" src=\"https:\/\/eamonimgbed.oss-cn-beijing.aliyuncs.com\/img\/202302261527845.png?x-oss-process=style\/screenshots\">\u56e0\u4e3a\u5d4c\u5408\u9f20\u4e2dES\u7ec6\u80de\u6765\u6e90\u7684KO-First allele\u53ef\u80fd\u8fdb\u5165\u751f\u6b96\u7cfb\u7ec6\u80de\uff0c\u4e5f\u53ef\u80fd\u6ca1\u6709\u8fdb\u5165\uff0c\u6240\u4ee5\u9700\u8981\u5bf9F1\u5c0f\u9f20\u8fdb\u884c\u57fa\u56e0\u578b\u9274\u5b9a\uff08genotyping)\u3002<\/li>\n\n\n\n<li>\u6211\u4eec\u60f3\u8981\u6784\u5efa\u7684\u662fCre\u63a7\u5236\u7684\u6761\u4ef6\u6027\u57fa\u56e0\u6572\u9664\u5c0f\u9f20\uff0c\u6240\u4ee5\u9700\u8981\u901a\u8fc7FLP\u5c06tm1a\u8f6c\u53d8\u4e3atm1c\u3002\u5c06F1\u6742\u5408\u5b50\u4e0e\u7eaf\u5408\u7684FLP\u5c0f\u9f20\u6742\u4ea4\u3002\u8fd9\u6837\uff0c\u6211\u4eec\u67091\/2\u7684\u6982\u7387\u5f97\u5230\u4e0b\u9762\u7684F2\u5c0f\u9f20\u3002<img loading=\"lazy\" decoding=\"async\" decoding=\"async\" alt=\"image-20230226152753480\" src=\"https:\/\/eamonimgbed.oss-cn-beijing.aliyuncs.com\/img\/202302261527874.png?x-oss-process=style\/screenshots\">\u53e6\u59161\/2\u662fwt\/wt\uff0c\u6240\u4ee5\u4ecd\u7136\u9700\u8981genotyping\uff0c\u786e\u5b9a\u4e24\u6761allele\u4e2d\u4e00\u6761\u662ftm1c\uff0c\u4e00\u6761\u662fWT\u3002\u7531\u4e8eKnockout first allele\u4e2dfrt\u95f4\u5220\u9664\u5e8f\u5217\u8f83\u957f\uff0c\u5e76\u4e14Flp\u5220\u9664\u6548\u7387\u7684\u9650\u5236\uff0c\u5728\u7b2c\u4e00\u4ee3\u83b7\u5f97\u7684\u6761\u4ef6\u6027\u6572\u9664\u5c0f\u9f20\uff08tm1c+\/-\uff0cFlp+\/-\uff09\u4e2d\uff0c\u53ef\u80fd\u4f1a\u51fa\u73b0tm1a allele\u3002\u7531\u4e8e\u5728\u8fd9\u4e00\u4ee3\u5c0f\u9f20\u4e2d\uff0c\u6bcf\u4e2a\u7ec6\u80de\u4e2d\u90fd\u5728\u53d1\u751fFlp\u5bf9Frt\u5e8f\u5217\u7684\u8bc6\u522b\u548c\u5207\u5272\u8fc7\u7a0b\uff0c\u65e0\u6cd5\u4fdd\u8bc1\u6bcf\u4e2a\u7ec6\u80de\uff08\u5c24\u5176\u662f\u751f\u6b96\u7ec6\u80de\uff09\u4e2d\u7684\u76ee\u7684\u5e8f\u5217\u90fd\u5b8c\u5168\u88ab\u5220\u9664\u3002\u5efa\u8bae\u7b2c\u4e00\u4ee3\u83b7\u5f97\u7684\u6761\u4ef6\u6027\u6572\u9664\u5c0f\u9f20\u5148\u4e0e\u91ce\u751f\u578b\u5c0f\u9f20\u4ea4\u914d\u4e00\u4ee3\uff0c\u83b7\u5f97tm1c+\/-\u4e14\u4e0d\u542bFLPeR\uff08Flp wt\/wt\uff09\u540e\u518d\u8fdb\u884c\u540e\u7eed\u7684\u4ea4\u914d\u3002\u8fd9\u6837\uff0c\u65b0\u4ea7\u751f\u7684\u5b50\u4ee3\u4ece\u53d7\u7cbe\u5375\u65f6\u671f\u5c31\u662f\u9776\u5e8f\u5217\u5b8c\u5168\u5220\u9664\u7684\u57fa\u56e0\u578b\uff0c\u4ece\u8fd9\u4e2a\u53d7\u7cbe\u5375\u53d1\u80b2\u800c\u6765\u7684\u6240\u6709\u7ec6\u80de\u90fd\u662f\u5b8c\u5168\u5220\u9664\u9776\u5e8f\u5217\u7684\u201c\u5e72\u51c0\u201d\u7684\u57fa\u56e0\u578b\u3002<\/li>\n\n\n\n<li>\u8fd9\u6837\u6211\u4eec\u5c31\u5f97\u5230\u4e86flox\u5c0f\u9f20\u7684\u6742\u5408\u5b50F2(flox\/+)\u3002F2\u81ea\u4ea4\uff0c\u540e\u4ee3\u67091\/4\u6982\u7387\u5f97\u5230\u7eaf\u5408\u7684flox\u5c0f\u9f20F3(flox\/flox)\u3002\u7efc\u4e0a\uff0c\u4ece\u5d4c\u5408\u4f53F0\u51fa\u53d1\uff0c\u7ecf\u8fc7\u4e09\u4ee3\u6742\u4ea4\uff0c\u6211\u4eec\u5c31\u80fd\u5f97\u5230\u9776\u57fa\u56e0\u7684\u7eaf\u5408flox\u5c0f\u9f20\u3010F1(tm1a\/wt), F2(tm1c\/wt), F3(tm1c\/tm1c)\u3011\u3002<img loading=\"lazy\" decoding=\"async\" decoding=\"async\" alt=\"image-20230226152839870\" src=\"https:\/\/eamonimgbed.oss-cn-beijing.aliyuncs.com\/img\/202302261528078.png?x-oss-process=style\/screenshots\">\u4e0a\u56fe\u4f7f\u7528\u4e86\u4e0eES\u7ec6\u80de\uff08BN\u80cc\u666f\uff09\u4e0d\u540c\u80cc\u666f\u7684FLP\u5c0f\u9f20C57BL\/6J\uff08\u7b80\u79f0BJ\uff09\uff0c\u5982\u679c\u540e\u7eed\u6211\u4eec\u60f3\u5229\u7528\u9057\u4f20\u80cc\u666f\u4e3aBJ\u7684\u5c0f\u9f20\u8fdb\u884c\u5b9e\u9a8c\uff0c\u5c31\u9700\u8981\u5728F2\u4ee3\u4e0e\u91ce\u751f\u578bBJ\u201c\u56de\u4ea4\u201d\u6765\u7eaf\u5316\u80cc\u666f\uff0c\u4e00\u822c\u8ba4\u4e3a\u56de\u4ea410\u4ee3\u4ee5\u4e0a\u53ef\u4ee5\u5f97\u5230\u7eaf\u51c0\u7684\u9057\u4f20\u80cc\u666f\u3002\u5728\u5df2\u7ecf\u786e\u5b9a\u751f\u6b96\u9057\u4f20\u7684\u60c5\u51b5\u4e0b\uff0c\u53ef\u4ee5\u76f4\u63a5\u5c06\u5d4c\u5408\u9f20\u4e0eFLPeR\u4ea4\u914d\uff0c\u8fd9\u4e2a\u4ea4\u914d\u7b56\u7565\u53ef\u4ee5\u8282\u7ea61\u4e2a\u6708\u5de6\u53f3\u3002<img loading=\"lazy\" decoding=\"async\" decoding=\"async\" alt=\"image-20230226152908752\" src=\"https:\/\/eamonimgbed.oss-cn-beijing.aliyuncs.com\/img\/202302261529260.png?x-oss-process=style\/screenshots\"><\/li>\n\n\n\n<li>\u5f97\u5230\u7eaf\u5408flox\u5c0f\u9f20\u540e\uff0c\u4e0eCre\u9f20\u6742\u4ea4\u5f97\u5230KO\u9f20\u3002KO\u9f20\u9274\u5b9a\uff1a<img loading=\"lazy\" decoding=\"async\" decoding=\"async\" alt=\"image-20230226152937883\" src=\"https:\/\/eamonimgbed.oss-cn-beijing.aliyuncs.com\/img\/202302261529856.png?x-oss-process=style\/screenshots\"><\/li>\n<\/ol>\n\n\n\n<hr class=\"wp-block-separator has-alpha-channel-opacity\" \/>\n\n\n\n<h1 class=\"wp-block-heading\">\u6ce8\u91ca<\/h1>\n\n\n\n<h2 class=\"wp-block-heading\">\u5c0f\u9f20\u54c1\u7cfb\u7684\u547d\u540d<\/h2>\n\n\n\n<p><\/p>\n\n\n\n<p><\/p>\n\n\n\n<p>\u200b<\/p>\n\n\n\n<h2 class=\"wp-block-heading\">\u540d\u8bcd\u89e3\u91ca<\/h2>\n\n\n\n<p><a href=\"https:\/\/www.modelorg.com\/portal\/article\/index\/id\/2369\/cid\/28.html\">\u5c0f\u9f20\u57fa\u7840\u77e5\u8bc6\u4f60\u77e5\u9053\u591a\u5c11\uff1f\u4ec0\u4e48\u662f\u8fd1\u4ea4\u7cfb\u5c0f\u9f20\uff1f\u54e8\u5175\u9f20\u5462\uff1f\u56de\u4ea4\u548c\u6742\u4ea4\uff1f|\u5357\u6a21\u751f\u7269 (modelorg.com)<\/a><\/p>\n\n\n\n<h2 class=\"wp-block-heading\">\u836f\u7269\u63a7\u5236\u7684\u6761\u4ef6\u6027\u89e6\u53d1Cre<\/h2>\n\n\n\n<p>\u200b<\/p>\n\n\n\n<p><\/p>\n\n\n\n<p>\u200b<\/p>\n\n\n\n<p><\/p>\n\n\n\n<h2 class=\"wp-block-heading\">loxP sites\u7684\u8bbe\u8ba1\u539f\u5219<\/h2>\n\n\n\n<ol class=\"wp-block-list\">\n<li>\u5728\u5185\u542b\u5b50\u533a\u57df\uff0c\u4e24\u4e2aloxP site\u4e4b\u95f4\u5305\u4e00\u4e2a\u6216\u51e0\u4e2a\u8d77\u91cd\u8981\u529f\u80fd\u7684Exon<\/li>\n\n\n\n<li>\u4e24\u4e2aloxP site\u5c3d\u53ef\u80fd\u8fd1\uff0c\u4ee5\u4fbf\u66f4\u597d\u5730\u88abCre\u91cd\u7ec4<\/li>\n<\/ol>\n\n\n\n<hr class=\"wp-block-separator has-alpha-channel-opacity\" \/>\n\n\n\n<p>\u601d\u8003\uff1a<\/p>\n\n\n\n<ol class=\"wp-block-list\">\n<li>\u88abCre\u91cd\u7ec4\u51fa\u53bb\u7684DNA\u7684\u540e\u8def\u662f\u4ec0\u4e48\uff1f<\/li>\n\n\n\n<li>loxP sites\u80fd\u8bbe\u8ba1\u5728\u5916\u663e\u5b50\u533a\u57df\u5417\uff1f<\/li>\n\n\n\n<li>Cre\u53d1\u6325\u4f5c\u7528\u540e\uff0c\u80fd\u8bf1\u5bfcmRNA decay\u5417\uff1f\uff08\u5e08\u5144\u7684qPCR\u7ed3\u679c\u786e\u5b9e\u4f4e\u4e86\uff09<\/li>\n<\/ol>\n\n\n\n<hr class=\"wp-block-separator has-alpha-channel-opacity\" \/>\n\n\n\n<p>\u53c2\u8003\u6750\u6599\uff1a<\/p>\n\n\n\n<ol class=\"wp-block-list\">\n<li><a href=\"https:\/\/www.modelorg.com\/portal\/article\/index\/id\/2371.html\">\u3010\u5c0f\u9f20\u5927\u5b66\u95ee\u3011\u5c0f\u9f20\u57fa\u56e0\u7f16\u8f91\u795e\u5668<\/a><\/li>\n\n\n\n<li><a href=\"https:\/\/zhuanlan.zhihu.com\/p\/110259963\">CRISPR\/Cas9\u548cES\u6253\u9776\u5b70\u4f18\u5b70\u52a3\uff1f\u600e\u6837\u9009\u62e9\u624d\u662f\u6700\u4f18\u89e3\uff1f<\/a><\/li>\n\n\n\n<li><a href=\"https:\/\/zhuanlan.zhihu.com\/p\/52596420\">\u3010\u5e72\u8d27\u3011Cre-loxP\u91cd\u7ec4\u9176\u7cfb\u7edf\u4ecb\u7ecd<\/a><\/li>\n\n\n\n<li><a href=\"https:\/\/zhuanlan.zhihu.com\/p\/364198192\">\u771f\u6ca1\u60f3\u5230\uff0c\u7528\u4e86\u300e\u5b83\u300f\u4e0d\u52301\u4e2a\u6708\u5c31\u62ff\u5230\u4e86\u57fa\u56e0\u6572\u9664\u5c0f\u9f20\uff01<\/a><\/li>\n\n\n\n<li><a href=\"https:\/\/www.jax.org\/news-and-insights\/2006\/may\/the-cre-lox-and-flp-frt-systems\">The Cre-lox and FLP-FRT systems<\/a><\/li>\n\n\n\n<li><a href=\"https:\/\/www.cyagen.com\/us\/en\/community\/technical-bulletin\/knockout-first.html\">KO-First: A Tool for Studying Gene Function Across the Mammalian Genome<\/a><\/li>\n\n\n\n<li>\u8d5b\u4e1a\u751f\u7269\u5199\u7684KO-First\u6d41\u7a0b\u200b<\/li>\n<\/ol>\n","protected":false},"excerpt":{"rendered":"<p>\u8fd9\u7bc7\u7b14\u8bb0\u7cfb\u7edf\u68b3\u7406\u4e86\u6784\u5efa\u57fa\u56e0\u6572\u9664\u9f20\u7684\u7ecf\u5178\u7b56\u7565\uff0c\u91cd\u70b9\u89e3\u6790\u4e86\u7ed3\u5408Cre-loxP\u4e0eFLP-FRT\u7cfb\u7edf\u7684\u201cKnockout-First\u201d\u65b9\u6cd5\u3002\u6587\u7ae0\u8be6\u7ec6\u9610\u8ff0\u4e86\u5176\u8bbe\u8ba1\u539f\u7406\u3001\u4ece\u5b8c\u5168\u6027\u6572\u9664\u5230\u6761\u4ef6\u6027\u6572\u9664\u7684\u7075\u6d3b\u8f6c\u6362\u673a\u5236\uff0c\u4ee5\u53ca\u5177\u4f53\u7684\u6784\u5efa\u6b65\u9aa4\u4e0e\u6742\u4ea4\u7b56\u7565\u3002<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[4],"tags":[],"class_list":["post-157","post","type-post","status-publish","format-standard","hentry","category-note"],"_links":{"self":[{"href":"https:\/\/fanyiming.life\/index.php?rest_route=\/wp\/v2\/posts\/157","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/fanyiming.life\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/fanyiming.life\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/fanyiming.life\/index.php?rest_route=\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/fanyiming.life\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=157"}],"version-history":[{"count":2,"href":"https:\/\/fanyiming.life\/index.php?rest_route=\/wp\/v2\/posts\/157\/revisions"}],"predecessor-version":[{"id":14179,"href":"https:\/\/fanyiming.life\/index.php?rest_route=\/wp\/v2\/posts\/157\/revisions\/14179"}],"wp:attachment":[{"href":"https:\/\/fanyiming.life\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=157"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/fanyiming.life\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=157"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/fanyiming.life\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=157"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}