Construction of stable Cdc25C knockout HeLa cell strains using CRISPR/Cas9 gene-editing system
10.7644/j.issn.1674-9960.2017.05.007
- VernacularTitle:利用CRISPR/Cas9系统构建HeLa细胞Cdc25C基因敲除稳定细胞株
- Author:
Yebao YAO
;
Guangfei WANG
;
Qingcai DONG
;
Cheng CAO
;
Xuan LIU
- Keywords:
CRISPR/Cas9;
Cdc25C;
HeLa cell;
cell cycle
- From:
Military Medical Sciences
2017;41(5):359-362
- CountryChina
- Language:Chinese
-
Abstract:
Objective To knockout the cell division cycle 25 homolog C(Cdc25C) gene in HeLa human cervical cancer cells and to construct HeLa Cdc25C gene knockout stable strains using clustered regularly interspaced short palindromic repeats(CRISPR)/Cas9 gene-editing system.Methods The sequence of the small guide RNA(sgRNA),which could specifically recognize the first exon of Cdc25C,was designed according to the target-designing rules of CRISPR/Cas9 for construction of eukaryotic recombinant expressional plasmids.After sequencing,the plasmid was transfected into HeLa cells.The stable Cdc25C-knocking out strains were screened through the stress of puromycin,and the knockout effect was detected by Western blotting.The cell cycle was analyzed by flow cytometry.Results The stable Cdc25C-knocking out strains were obtained.Moreover,the gene′s knockout obviously delayed the progression of G2/M phase.Conclusion The HeLa Cdc25C gene knockout stable strain is successfully built using CRISPR/Cas9 system,facilitating studies on the function of Cdc25C and the mechanism of carcinogenesis.
