利用CRISPR_Cas9技术构建TGFBI敲除的Met-5A稳定细胞株  

Construction of stable human Met-5A cells in which TGFBI is knockout by CRISPR_Cas9 technology

作　　者：杨杰[1] 郑晋南 兰亚佳[1] 张勤[1] YANG Jie;ZHENG Jin-nan;LAN Ya-jia;ZHANG Qin(Department of Environmental Health and Occupational Medicine,West China School of Public Health and West China Fourth Hospital,Sichuan University,Chengdu,Sichuan 610041,China)

机构地区：[1]四川大学华西公共卫生学院/四川大学华西第四医院

出　　处：《现代预防医学》2019年第15期2837-2841,共5页Modern Preventive Medicine

基　　金：四川省卫生和计划生育委员会科研课题2016年普及应用项目(编号:16PJ261)

摘　　要：目的利用CRISPR_Cas9基因编辑技术构建TGFBI敲除的Met-5A稳定细胞株,为进一步深入研究TGFBI低表达在人膜间皮细胞恶性转化过程中的功能角色提供技术支持。方法根据CRISPR_Cas9靶点设计原则针对TGFBI基因第4号外显子设计2对sgRNA(小向导RNA),退火形成双链,连接至Bsmbl酶切后的载体质粒lentiCRISPRv2(命名为lentiCRISPRv2-T1和lentiCRISPRv2-T2),经感受态细胞扩增后提取质粒并测序验证。提取和纯化正确插入目的片段的重组质粒,经慢病毒包装并转染Met-5A细胞,嘌呤霉素筛选阳性克隆。提取细胞及上清液总mRNA和蛋白,使用实时定量聚合酶链反应(RT-qPCR)和免疫印迹实验(Western blot)检测慢病毒系统转染后TGFBI mRNA和蛋白表达水平的变化。结果经基因测序后证实lentiCRISPRv2-T1、lentiCRISPRv2-T2重组载体质粒均正确插入了sgRNA序列;慢病毒系统转染Met-5A细胞后,v2-T1、v2-T2组TGFBI mRNA相对表达水平相比对照组明显降低(P<0.05),且v2-T2组TGFBI mRNA相对表达水平低于v2-T1组(P<0.05);v2-T1组TGFBI蛋白表达量下降为对照组的0.03倍,在v2-T2细胞中几乎检测不到TGFBI蛋白。结论构建了针对TGFBI第4号外显子的两组sgRNA-lentiCRISPRv2敲除载体;成功获得了TGFBI敲除的Met-5A稳定细胞株,为进一步研究TGFBI低表达在恶性胸膜间皮瘤发生发展过程中的分子机制奠定了基础。Objective To obtain stable human Met-5 A cells in which TGFBI gene is knockout by CRISPR_Cas9 gene-editing technology,and to provide technical support for further research on the low expression of TGFBI in the malignant transformation of human mesothelial cells.Methods Two pairs of sgRNA(small guide RNA)targeting the fourth exon of TGFBI were designed according to the guideline of CRISPR_Cas9 system.The sgRNA was then ligated into the Bsmbl-digested lentiCRISPRv2(named lentiCRISPRv2-T1 and lentiCRISPRv2-T2),and the recombinant plasmid was verified by sequencing technique after amplification of competent cells.The plasmid which was correctly inserted into the target fragment was extracted,purified,packaged by lentivirus,transfected into Met-5 A cellsand selected by puromycin.In addition,total RNA and protein of Met-5 A cells and supernatant were extracted.Real time-quantitative polymerase chain reaction(RT-qPCR)and Western Blot assays were performed to detect the altered expression level of TGFBI after lentivirus system transfection.Results The two pairs of sgRNA were correctly inserted into the recombinant plasmid lentiCRISPRv2-T1 and lentiCRISPRv2-T2.When transfected with lentivirus system,the relative expression level of TGFBI mRNA in v2-T1 and v2-T2 groups was significantly lower than that in the control group(P<0.05),and the relative expression level of TGFBI mRNA in v2-T2 group was lower than that in v2-T1 group(P<0.05).The expression level of TGFBI protein in v2-T1 group was decreased to 0.03 times of control group,and TGFBI protein was hardly detected in v2-T2 group.Conclusion Two groups of sgRNA-lentiCRISPRv2 vectors targeting the fourth exon of TGFBI are constructed.The human Met-5 A cells in which TGFBI gene is knockout are successfully obtained,laying a foundation for further research on the molecular mechanism of low expression of TGFBI in the development and progression of malignant pleural mesothelioma.

关 键 词：CRISPR/Cas9 TGFBI Met-5A细胞 恶性胸膜间皮瘤 

分 类 号：R113[医药卫生—公共卫生与预防医学]