应用CRISPR/Cas9技术构建MAVS基因敲除的ZR-751乳腺癌细胞株

目的 运用成簇的规律间隔短回文重复序列(CRISPR)/Cas9基因编辑技术,构建线粒体抗病毒信号蛋白(MAVS)基因敲除的ZR-751乳腺癌细胞株,研究MAVS对细胞生长的影响.方法 针对MAVS基因第一外显子设计小向导RNA(sgRNA),构建pX459-sgRNA重组质粒.然后利用嘌呤霉素筛选ZR-751 MAVS基因敲除的阳性克隆,Western印迹检测MAVS基因敲除情况,提取克隆基因组进行测序鉴定.平板克隆实验检测MAVS被敲除后对细胞增殖的影响,再利用MTS实验检测在DFX培养基刺激下,MAVS对细胞死亡的影响.结果 Western印迹结果说明ZR-751细胞株内MAVS已完全敲除;且在凋亡诱导剂DFX刺激下,MAVS被敲除后促进细胞增殖.结论 利用CRISPR/Cas9系统成功构建了MAVS基因敲除的ZR-751乳腺癌细胞株,初步实验提示MAVS抑制乳腺癌细胞生长,为后续研究MAVS在肿瘤中的功能奠定了基础.

Construction of MAVS knockout ZR-751 stable strain by CRISPR/Cas9 technology

Objective To construct mitochondrial antiviral-signaling protein ( MAVS ) knockout ZR-751 breast neoplasms cells using CRISPR/Cas9 genome engineering technology , and study the effect of MAVS on cell proliferation . Methods Small guide RNA ( sgRNA ) was designed by targeting the first exon of MAVS gene and the pX 459-sgRNA recombinant eukaryotic expressional plasmid was constructed .Puromycin was used to screen monoclonal cells which stably knocked out MAVS gene .The knockout effect was measured by Western blotting .Cellular proliferation rates were detected by colony-forming assay when MAVS gene was knockout .The MTS assay was designed to detect the effect of MAVS on cell proliferation under DFX stimulus .Results The result of Western blotting suggested that no MAVS protein was detected in the MAVS gene knockout stable ZR-751 cells,showing that MAVS gene was knocked out completely .Proliferation became faster when MAVS was knocked out .MAVS promoted cell death under DFX stimulus .Conclusion The MAVS knockout ZR-751 stable cells have been constructed using CRISPR/Cas9 system.The preliminary experimental results show that MAVS inhibits breast cancer cell proliferation , which will facilitate studies on the function of MAVS in tumors in the future .