| 张淑静 高誉珊 孙红梅 许红 王媛媛 刘谦 胡雨 吴莹 和欣 龚小钢.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[J].现代生物医学进展英文版,2015,15(23):4401-4405. |
| 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 MousePINK1 Gene and Screening in NIH3T3 Cells |
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| DOI： |
| 中文关键词: PINK1-siRNA 慢病毒载体 NIH3T3 细胞 帕金森病 |
| 英文关键词: PINK1-siRNA Lentiviral vector NIH3T3 cell Parkinson's disease |
| 基金项目: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） |
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| 中文摘要: |
| 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。 |
| 英文摘要: |
| 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 293T cell to conform the drops of Lentivirus
particles. Then the virus particles were transfected into the NIH3T3 cells, using qRT-PCR to verify the PINK1-siRNA 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 mRNA expression, which provides a molecular biology technology for
further study the pathogenesis of Parkinson's disease. |
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