摘要
目的 :利用CRISPR/Cas9技术构建OSBPL2基因敲除的稳定HeLa细胞株。方法 :设计3个单导向RNA(singleguide RNA,sg RNA),分别靶向OSBPL2基因的第2、3和5外显子,以PGK1.1为载体,构建出3个重组真核表达载体。分别转染HeLa细胞后,使用嘌呤霉素进行阳性细胞筛选,Cruiser TM敲除检测实验和测序共同检验载体靶向效率。选出靶向效率最高的质粒转染HeLa细胞,进行单克隆细胞培养,最后用Western blot鉴定敲除效果。结果:sg RNA正确插入到PGK1.1载体,靶向第2外显子的重组载体转染HeLa细胞并筛选单克隆后,细胞未检测出OSBPL2蛋白的表达。结论:敲除OSBPL2基因的稳定HeLa细胞株构建成功。
Objective：To apply CRISPR / Cas9 technology to construct stable OSBPL2 gene knockout HeLa strain. Method：Three single-guide RNA（sg RNA） targeting respectively to exon 2,3,5 of OSBPL2 gene were designed, then 3 recombinant eukaryotic expressional plasmids by the carrier of PGK1.1 were constructed. After transfection to HeLa cell respectively, puromycin was performed to screen positive cells and then cruiserTMgene knockout detection in combination with the sequencing were used to analyze targeting effect of the three plasmids. HeLa cells with the plasmid of the optimal targeting effect continued to screen monoclonal cell. The knockout effect was measured by Western blot. Results： Sg RNAs were correctly inserted into the recombinant plasmids, OSBPL2 protein was undetected in HeLa cell after transfection with plasmid targeting to exon 2 and screening of monoclonal cell. Conclusion：Stable OSBPL2 gene knock-out HeLa strain can be successfully built.
出处
《南京医科大学学报（自然科学版）》
CAS
CSCD
北大核心
2016年第9期1072-1078,共7页
Journal of Nanjing Medical University(Natural Sciences)
基金
国家自然科学基金(31171217
31571302)
