利用改良的CRISPR/Cas9基因编辑系统构建HeLa细胞SND1基因敲除稳定株

Construction of He La SND1 knockout gene stable strain by using modified CRISPR/Cas9 gene editing system

目的:利用改良的CRISPR/Cas9基因编辑系统敲除He La细胞中SND1基因,构建He La细胞SND1基因敲除稳定株。方法:设计一对特异性识别SND1基因第二个启动子的上下游sg RNA,以PX462质粒为载体,构建出一对重组真核表达质粒。酶切和测序鉴定后,将一对重组质粒共同转染进入He La细胞中,使用嘌呤霉素进行阳性细胞筛选,挑取单克隆细胞进行培养。最后用Western Blot鉴定敲除效果。结果:sg RNA正确插入到PX462质粒载体中,转染并筛选单克隆后的细胞中没有SND1蛋白的表达。结论:成功构建出He La细胞SND1基因敲除稳定株。

Objective：To apply modified CRISPR/Cas9 gene editing system to knock out the SND1 gene in HeLa cell and construct HeLa SND1 gene knockout stable strain. Methods：A pair of sgRNAs that could specially identify the upstream and downstream of SND1 gene second promoter were designed, then a recombinant eukaryotic expressional plasmid by the carrier of PX462 was constructed. After enzyme digestion and sequencing, a pair of recombinant plasmids into HeLa cell were co-transfected, then puromycin was used to screen positive cell and the monoclonal cell was developed. The knockout effect was measured by western blotting. Results：sgRNA was correctly inserted into the PX462 recombinant plasmid, and SND1 protein was undetected in HeLa cell after transfection and screening of monoclonal cell. Conclusion：HeLa SND1 gene knockout stable strain can be successfully built.