DAI Xin, ZHAO Junli, YANG Peiyan, XIA Haibin*
（School of Life Sciences, Shaanxi Normal University, Xi′an 710119, Shaanxi, China）
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.
KeyWords:
Glutaredoxin 3(GLRX3); CRISPR/Cas9; gene knockout; genome editing
