基于CRISPR/Cas9系统建立SNX11基因敲除A549细胞系

Construction of A SNX11-knockout A549 Cell Line Based on the CRISPR/Cas9 System

为了建立SNX11基因稳定敲除A549细胞系,本研究通过构建针对人SNX11基因的特异性打靶慢病毒载体,将该慢病毒感染A549细胞系,使用嘌呤霉素对慢病毒感染阳性的A549细胞进行筛选,利用梯度稀释法获得单细胞并扩增培养,提取细胞基因组DNA,PCR扩增后进行测序验证,同时提取细胞蛋白后利用Western Blot方法进行蛋白敲除验证,最后进行细胞活性检测和脱靶效应评估。最后,本研究获得了一株SNX11基因敲除A549细胞系;通过脱靶效应评估,结果显示最可能的20个脱靶位点均不存在脱靶现象;该基因敲除对细胞增殖活性没有影响。本研究成功构建了一株SNX11基因敲除A549细胞系,为SNX11蛋白的功能研究建立了细胞模型。

We wished to establish an A549 cell line with knockout of the SNX11 gene. We constructed a lentivirus-specific vector targeting human SNX11. Then,we infected A549 cell lines with the lentivirus,screened A549 cells positive for lentivirus infection with puromycin,obtained single cells by gradient dilution,and amplified the culture. Genomic DNA was extracted and amplified by polymerase chain reaction for sequencing verification. Simultaneously,proteins were extracted and verified by western blotting. Cell activity was detected and off-target effects evaluated. Finally,an SNX11-knockout A549 cell line was obtained. There were no off-target effects in the 20 most likely sites. Gene knockout had no effect on cell proliferation. We constructed an SNX11-knockout A549 cell line,which established a cell model for functional study of SNX11protein.