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.
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)
出处
《生物技术通讯》
CAS
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)
