用聚乙烯亚胺反向转染siRNA表达盒进行RNA干扰的研究

Efficient RNA Interference Through Polyethylenimine Mediated Reverse Transfection of siRNA Expression Cassettes

目的:建立适用于大规模RNA干扰(RNAi)筛选的siRNA生成与导入技术。方法:以绿色荧光蛋白(GFP)为模型,用PCR方法生成了包含U6启动子、互补的反义链和正义链以及终止序列的特异性siRNA表达盒(SEC),对聚乙烯亚胺(PEI)介导的转染SEC的方法及其效率进行研究。结果:PEI对细胞的毒性呈剂量依赖关系,当PEI与DNA的N/P=7.53,即PEI与DNA等量时,转染效率达到最高。PEI与脂质体LipofectAMINETM2000的转染效率无显著差异(P>0.05),对SEC的转染效率显著高于质粒载体(P<0.01)。反向转染的转染效率显著高于常规转染(P<0.01)。利用PEI将表达GFP特异性siRNA的SEC反向转染可稳定表达GFP的HeLa细胞株(HeLa-EGFP),荧光染色和Western印迹检测均表明可显著抑制GFP的表达。结论:PEI介导的SEC反向转染具有简便、快捷、经济的优点,可满足大规模RNAi筛选的需要。

Objective： To develop a comprehensive siRNA preparation and introduction technique that can be adapted to large-scale RNA interference（RNAi） screening. Methods： The practibility of polyethylenimine（PEI） mediated siRNA expression cassettes（SEC） transfection was evaluated by using green fluorescent protein（GFP） as a target. SEC containing U6 promoter, complementary anti-sense, sense strands and terminator sequence in sequence were prepared by PCR and the methodology and efficiency of transfection were investigated. Results： The cytotoxicity of PEI was dose-dependent. The transfection efficiency peaked when the N/P=7.53, i.e. the same amount PEI was mixed with DNA. There is no significant difference in transfecion efficeincy between PEI and LipofectAMINE^TM 2000（P〉0.05）. The transfecion efficiency of SEC mediated by PEI was significant higher than that of plasmid vector（P〈0.01）. Reverse transfection was more effective than routine transfection （P〈O.01）. Both fluorescence staining and Western blotting showed apparent suppression of GFP expression through the PEI mediated reverse transfection of SEC expressing siRNAs targeting GFP in HeLa-GFP, a HeLa ceU line which stably expresses the GFP protein. Conclusion： The PEI mediated reverse transfection of SEC is a money-saving and convenient approach that can be applied to large-scale RNAi screening.