利用CRISPR/Cas9技术构建ATF4基因敲除的HepG2细胞株

Construction of ATF4 knockout HepG2 cell line by CRSPR/Cas9

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。

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.