利用CRISPR/Cas9技术构建ATF4基因敲除的HepG2细胞株

摘要

Objective:To construct ATF4 knockout HepG2 cells by using CRSPR/Cas 9 genome engineering technology.Methods:Two pairs of gRNAs targeting the ATF4 genes Exon 1 and 2 were designed for the functional domains of the ATF4 gene.The recombinant plasmid PX459-gRNA was constructed and transformed into competent cells DH5α.The recombinant plasmid was screened and sequenced.The effectiveness of the designed gRNA was confirmed by sequencing.PX459-ATF4-gRNA was transfected into HepG2 cells.After cloning and sequencing,The ATF4 knockout HepG2 cells were obtained and the expression of ATF4 was detected by western blotting.Results:The knockout plasmid was successfully constructed.Compared with negative control group,ATF4 gene was not expressed in the transfected PX459-ATF4-gRNA plasmid group.Conclusion:The ATF4 knockout HepG2 cell line was successfully constructed by using CRSPR/Cas 9 system,which lays the foundation for further studies of the effect and mechanisms of ATF4 in liver cancer.%目的:运用CRISPR/Cas9基因编辑技术,建立ATF4基因敲除的HepG2细胞株.方法:针对ATF4基因作用的功能区域,设计靶向ATF4基因Exon 1(外显子1)和Exon 2(外显子2)的两对gRNA.构建PX459-ATF4-gRNA重组质粒并转化DH5α感受态细胞,筛选出重组子后进行测序,通过测序验证所设计的gRNA的有效性.将PX459-ATF4-gRNA质粒转染到HepG2细胞中,挑选单克隆细胞,提取DNA,进行PCR检测与基因测序,获得敲除ATF4基因的细胞株.通过western blotting检测筛选出来的HepG2细胞ATF4基因的敲除效果.结果:菌落PCR与菌液测序结果显示PX459-ATF4-gRNA载体构建成功;PCR扩增电泳与基因测序检测显示成功构建成了ATF4基因敲除的HepG2细胞;western blotting检测结果显示单克隆2号ATF4蛋白不表达,单克隆3号仍表达ATF4蛋白.结论:成功构建ATF4基因敲除的HepG2细胞,为后续ATF4在肝癌中的作用机制和功能研究奠定基础.