【摘要】 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。更多还原

【Abstract】 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 LipofectAMINETM 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<0.01. 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 cell 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.更多还原