陕西师范大学学报（自然科学版）2017，Vol.45Issue(1)：71-77,7.DOI:10.15983/j.cnki.jsnu.2017.01.315
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
摘要
Abstract
To construct GLRX3-KO HEK293 cell line,four sgRNAs targeting the Exon5 of GLRX3 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.Subsequently,a monoclonal GLRX3 knockout cell line was isolated through puromycin screening and cell dilution cloning and sequence analysis indicated that both GLRX3 alleles contained frameshift mutation.The cell line was further confirmed at the expression level of GLRX3 protein by immunoblotting.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Key words
Glutaredoxin 3(GLRX3)/CRISPR/Cas9/gene knockout/genome editing分类
医药卫生引用本文复制引用
代鑫,赵俊丽,杨沛艳,夏海滨..利用CRISPR/Cas9技术构建GLRX3基因敲除的HEK293细胞系[J].陕西师范大学学报（自然科学版）,2017,45(1):71-77,7.基金项目
国家自然科学基金(81272543,31470058,81301957) （81272543,31470058,81301957）
陕西省自然科学基金(2014JZ005,2014JM4113) （2014JZ005,2014JM4113）
