小鼠TSC21基因敲除打靶载体的构建和胚胎干细胞同源重组克隆的鉴定.PDFVIP

Journal of Zhengzhou University(Medical Sciences)　 Jul.　 2018　 Vol.53　 No.4 ·511· doi:10.13705/ j.issn.1671-6825.2017.12.046 小鼠TSC21基因敲除打靶载体的构建和胚胎干细胞同源重组 克隆的鉴定 殷正伟,王朝亮,张天标,杨　 超,张卫星 郑州大学第一附属医院泌尿外科 郑州450052 关键词　 TSC21;基因敲除;打靶载体;同源重组;小鼠 + 中图分类号　 R698 .2 摘要　 目的:构建小鼠TSC21基因敲除打靶载体,电转胚胎干细胞(ES 细胞)并筛选同源重组阳性克隆。 方 法:订购含TSC21基因的129 BAC克隆,PCR扩增2对同源臂,利用PL253、PL452质粒进行目的片段的连接和同源 重组,构建打靶载体并电转ES细胞,利用Southern blot方法鉴定同源重组阳性ES细胞克隆。 结果:构建了TSC21 基因敲除打靶载体,经过限制性内切酶鉴定及DNA测序鉴定,TSC21基因敲除打靶载体构建成功。 Southern blot结 果显示成功筛选到打靶序列同源重组阳性ES细胞克隆。 结论:TSC21基因敲除打靶载体构建成功并筛选到同源 重组阳性ES细胞克隆。 Construction of mouseTSC21geneknockouttargetingvector andidentifi- cation of homologous recombination clone of embryonic stem cells YIN Zhengwei,WANG Chaoliang,ZHANG Tianbiao,YANG Chao,ZHANG Weixing Department of Urology,the FirstAffiliated Hospital,Zhengzhou University,Zhengzhou450052 Keywords　 TSC21;gene knockout;targeting vector;homologous recombination;mouse Abstract　 Aim:To construct mouse TSC21gene knockout targeting vector,transform the targeting vector into em- bryonic stem(ES) cells using electroporate method,and screen homologous recombination occurred ES cells.Methods: 129 BAC clones which contain TSC21gene was ordered,two pairs of homologous arms were amplified by PCR method. PL253,PL452 plasmidwereusedforligationandhomologousrecombinationbetweendifferentfragments.Targetingvector was constructed and transformed into ES cells by electroporate method.Southern blot was used to identify homologous re- combination occurred EScell clones.Results:TSC21gene knockouttargetingvectorwasconstructed. The correct struc- ture of thetargetingvectorwasconfirmedbytheresultsofrestrictionendonucleaseenzymedigestionandDNA sequencing analysis. Southernblotresult showedthat homologousrecombination-positive EScell clonesexisted.Concl