慢病毒介导的小鼠PINK1基因RNAi载体的构建及在NIH3T3细胞中的筛选  

Construction of a Lentivirus-Mediated RNAi Vector Targetting Mouse PINK1 Gene and Screening in NIH3T3 Cells

作　　者：张淑静[1] 高誉珊[1] 孙红梅[1] 许红[1] 王媛媛[1] 刘谦[2] 胡雨[1] 吴莹[1] 和欣[1] 龚小钢 

机构地区：[1]北京中医药大学,北京100029 [2]首都医科大学附属北京同仁医院,北京100730

出　　处：《现代生物医学进展》2015年第23期4401-4405,共5页Progress in Modern Biomedicine

基　　金：北京中医药大学自主课题(2013-JYBZZ-JS-123);国家自然科学基金项目(30873335)

摘　　要：目的:构建筛选靶向特异PINK1-siRNA慢病毒表达载体,感染小鼠胚胎细胞(NIH3T3)验证该病毒载体的敲减效率,为研究帕金森病的发病机制奠定基础。方法:构建2对靶向小鼠PINK1-siRNA序列(KD1和KD2),将这2对序列连接在GV118上,将重组载体和病毒包装的辅助质粒共转染293 T细胞,获得慢病毒颗粒,再将该病毒颗粒转染入NIH3T3细胞,用qRT-PCR验证细胞内PINK1 mRNA表达水平以验证敲减效果。结果:筛选出可以用于后续实验的高效靶向KD2序列,并成功转染到小鼠NIH3T3细胞中,在感染复数(MOI)为50时,KD2的沉默效果最好,敲减率达到66.4%。结论:成功构建了高效靶向小鼠PINK1-siRNA慢病毒载体,其可稳定转染小鼠NIH3T3细胞,可高效抑制PINK1 mRNA的表达。为下一步利用其感染神经细胞或注入动物脑内,进一步研究PINK1基因在帕金森病发病过程中的作用环节和机制提供了分子生物学的技术基础。Objective： To construct lentiviral vector of RNA interference（RNAi）targeting mouse PINK1 gene, to infect mouse embryonic cells（NIH3T3） to test the virus＇ interfering efficiency, and to lay a foundation for studying the pathogenesis of Parkinson＇s disease. Methods： Two PINK1-siRNA sequences on the mouse（KD1 and KD2） were constructed and connected the two sequences to the GV118, then the recombinant virus and packaging auxiliary plasmids were co-transfected 293 T cell to conform the drops of Lentivirus particles. Then the virus particles were transfected into the NIH3T3 cells, using q RT-PCR to verify the PINK1-si RNA recombinant expression vectors＇ knock down effection on the inhibition of PINK1 mRNA expression level. Results： An efficient sequence for PINK1-siRNA was built, and the target KD2 can be used for the following experiments, the efficient KD2 sequence was packed and successfully transfected into mouse NIH3T3 cells, when the MOI is 50, KD2 had the highest interfering efficiency, and the interfering efficiency of lentivirus was 66.4%. Conclusion： The efficient mouse PINK1-siRNAlentiviral vector was successfully built, and it can be stably transfected mouse NIH3T3 cells and efficiently suppress PINK1 m RNA expression, which provides a molecular biology technology for further study the pathogenesis of Parkinson＇s disease.

关 键 词：PINK1-siRNA 慢病毒载体 NIH3T3细胞 帕金森病 

分 类 号：Q95-3[生物学—动物学] Q78