论文部分内容阅读
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p X459-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。
Objective To construct mitochondrial anti-viral signal protein (MAVS) knockout ZR-751 breast cancer cell lines using clustered regularly spaced short palindrome repeat (CRISPR) / Cas9 gene editing techniques to study the effect of MAVS on cell growth. Methods Based on the first exon of MAVS gene, a small guide RNA (sgRNA) was designed to construct pX459-sgRNA recombinant plasmid. Then the positive clones of ZR-751 MAVS knockout gene were screened by puromycin, the MAVS gene knockout was detected by Western blot, and the cloned genome was extracted for sequence identification. The effect of MAVS knockdown on cell proliferation was tested by plate clone assay. The effect of MAVS on cell death was tested by MTS assay in DFX medium. Results Western blotting results showed that MAVS in ZR-751 cells had been completely knocked out. Under the stimulation of apoptosis inducer DFX, MAVS knocked out and promoted cell proliferation. Conclusion MAVS knockout ZR-751 breast cancer cell line was successfully established by CRISPR / Cas9 system. Preliminary experiments suggested that MAVS could inhibit the growth of breast cancer cells, which laid the foundation for the further study on the function of MAVS in tumor.