Vasorin基因稳定敲除HepG2细胞株的建立及其生物学功能研究  

Establishment and Biological Function of Vasorin (VASN) Gene Stably Knockout HepG2 Cell Lines

作　　者：耿介 王超男 李少华 丁红梅 李慧 黄皑雪 李洁 李达 白琛俊 张坦 董洁 邵宁生 GENG Jie;WANG Chao-Nan;LI Shao-Hua;DING Hong-Mei;LI Hui;HUANG Ai-Xue;LI Jie;LI Da;BAI Chen-Jun;ZHANG Tan;DONG Jie;SHAO Ning-Sheng(Institute of Military Cognitive and Brain Sciences,Academy of Military Medical Sciences,Beijing 100850,China)

机构地区：[1]军事医学研究院军事认知与脑科学研究所,北京100850

出　　处：《生物技术通讯》2018年第2期162-167,共6页Letters in Biotechnology

基　　金：国家自然科学基金(81572846;31570817)

摘　　要：目的:利用CRISPR/Cas9技术建立人Vasorin(VASN)基因稳定敲除的HepG2细胞株,并研究VASN蛋白对细胞增殖和迁移等生物学功能的影响。方法:根据人VASN序列设计向导RNA,将其克隆至表达载体pCas-Guide,获得质粒pCas-gRNA;PCR扩增VASN编码基因左、右同源臂及潮霉素B抗性基因,通过重叠PCR将三者拼接为一条片段,并克隆至载体pBackZero-T,获得质粒pBackZero-T-VASN;上述2种重组质粒共转染HepG2细胞,经潮霉素B筛选,通过基因组PCR、RT-q PCR和Western印迹实验检测VASN基因是否被敲除。利用CCK-8法和Transwell法检测VASN稳定敲除细胞株的增殖和迁移能力。结果:向导RNA表达质粒pCas-gRNA和DNA供体质粒pBackZero-T-VASN构建成功;基因组PCR结果显示,潮霉素B基因正确重组至VASN基因中;RT-PCR显示VASN m RNA水平显著降低,Western免疫印迹结果显示VASN蛋白表达水平显著降低;VASN稳定敲除细胞株的增殖和迁移能力比野生型细胞明显减弱。结论:构建了pCas-gRNA和pBackZero-T-VASN质粒,获得了VASN缺失的细胞株,验证了VASN蛋白参与细胞的增殖和迁移过程,为深度探讨VASN的生物学功能及其分子机制奠定了基础。Objective:To establish the HepG2 cell line with human vasorin(VASN)gene stably knockout by CRISPR/Cas9 technology,and address biological functions of VASN protein.Methods:The small guidance RNA of VASN was cloned into the eukaryotic expression vector and the recombinant pCas-gRNA was acquired.The left,right homologous arms of VASN gene were amplificated and combined by PCR,then cloned into pBackZero-T vector to obtain the recombinant pBackZero-T-VASN.The two recombinant plasmids were transfected into HepG2 cells and screened by hygromycin B,then the expression of VASN was assayed by genomic PCR,RT-qPCR and Western blotting.The proliferation and migration ability of the VASN stably knockout cell lines were detected by CCK-8 and transwell assay.Results:The plasmids pCas-gRNA and pBackZero-T-VASN were constructed successfully.The result of genomic PCR showed that the hygromycin B gene was correctly reorganized into the VASN gene.The result of RT-qPCR showed that the level of VASN mRNA was significantly reduced,and the result of Western blotting showed a significant decrease in the protein expression level.The proliferation and migration of the VASN stably knockout cell lines were significantly decreased than those of wild-type cells.Conclusion:The plasmids pCas-gRNA and pBackZero-T-VASN were constructed successfully.The VASN knockout cell line was established successfully.And it was suggested that the VASN protein was involved in cell proliferation and migration process.These results might lay a foundation for in-depth researches on VASN biological functions.

关 键 词：CRISPR/Cas9技术 基因敲除 Vasorin(VASN)基因 细胞增殖 细胞迁移 

分 类 号：Q78[生物学—分子生物学]