利用CRISPR/Cas9技术构建GLRX3基因敲除的HEK293细胞系  

Construction of GLRX3-knockout HEK 293 cell line by CRISPR/Cas9 technology

作　　者：代鑫[1] 赵俊丽[1] 杨沛艳[1] 夏海滨[1] 

机构地区：[1]陕西师范大学生命科学学院,陕西西安710119

出　　处：《陕西师范大学学报（自然科学版）》2017年第1期71-77,共7页Journal of Shaanxi Normal University：Natural Science Edition

基　　金：国家自然科学基金(81272543,31470058,81301957);陕西省自然科学基金(2014JZ005,2014JM4113)

摘　　要：为了构建GLRX3(Glutaredoxin 3)敲除的HEK293细胞系,根据该蛋白结构特性设计了靶向于GLRX3基因Exon5的4个sgRNA,并通过T7E1检测确认了所设计sgRNA的有效性。将携带Cas9及靶向hGLRX3-Exon5的sgRNA1的打靶载体转染HEK293,通过药物Puromycin和细胞克隆化筛选出稳定GLRX3基因敲除的HEK293细胞株,并通过测序确定GLRX3两个等位基因均发生移码突变。通过免疫印迹在蛋白表达水平确认了该基因的敲除。利用CRISPR/Cas9技术成功构建了GLRX3敲除的HEK293细胞株,其为探索GLRX3的功能和作用机制提供了有效的细胞模型。To construct GLRX3-KO HEK293 cell line, four sgRNAs targeting the Exon5 of GL- RX3 gene were designed according to the characteristics of GLRX3 protein spatial structure, and the biological activities of these sgRNA were confirmed by T7E1 assay. Then targeting vector carrying Cas9 with sgRNA1 for hGLRX3-Exon5 was transfected into HEK293 cells. Subsequent- ly, a monoclonal GLRX3 knockout cell line was isolated through puromycin screening and cell di- lution cloning and sequence analysis indicated that both GLRX3 alleles contained frameshift muta- tion. The cell line was further confirmed at the expression level of GLRX3 protein by immunob- lotting. In summary, a GLRX3-knockout HEK293 cell line was successfully generated by using CRISPR/Cas9 system, which would provide a powerful cell model for studying the functions of GLRX3 and related mechanisms involved.

关 键 词：GLRX3 CRISPR/Cas9系统 基因敲除 基因编辑 

分 类 号：R393[医药卫生—基础医学]