利用CRISPR/Cas9系统构建HeLa细胞Cdc25C基因敲除稳定细胞株

Construction of stable Cdc25C knockout HeLa cell strains using CRISPR/Cas9 geneediting system

目的使用CRISPR/Cas9基因编辑系统敲除人类宫颈癌细胞HeLa中的细胞分裂周期蛋白25同源蛋白C(cell division cycle 25 homolog C,Cdc25C)基因,构建Cdc25C基因稳定敲除细胞株。方法根据CRISPR/Cas9靶点设计规则,设计特异性识别Cdc25C基因第一外显子相关序列的上下游小向导RNA(small guide RNA,sgRNA),构建真核重组表达质粒。测序鉴定后,将重组质粒转染至HeLa细胞中,用嘌呤霉素(puromycin)抗性筛选稳定敲除Cdc25C基因细胞株,再用免疫印迹方法鉴定细胞Cdc25C敲除效果。最后用流式细胞仪检测基因敲除对细胞周期的影响。结果筛选出稳定敲除Cdc25C基因细胞株,且Cdc25C敲除显著影响G2/M期进程。结论利用CRISPR/Cas9技术成功获得了内源Cdc25C基因敲除细胞株,为研究Cdc25C在细胞周期进程的功能以及相关癌症的发生奠定了基础。

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.