构建RNA干扰载体特异性抑制绿色荧光蛋白表达的研究

Construction of a siRNA expressing plasmid by PCR method that reduce EGFP expression in COS-7 cells efficiently

目的:利用PCR构建RNA干扰质粒载体的方法,促进RNA干扰技术的广泛开展。方法:PCR方法获得小鼠U6+27启动子序列,以及干扰绿色荧光蛋白(EGFP)表达的siRNA的相应DNA模板序列。将其序列克隆入pUC19,获得RNA干扰载体pU6-siEGFP。将pU6-siEGFP及pcDNA3.1/EGFP共转导COS-7细胞,观察EGFP表达情况。结果:所构建的pU6-siEGFP能够成功干扰COS-7细胞中EGFP的表达。结论:PCR方法成功构建了RNA干扰载体,为RNA干扰技术在更多实验室的广泛开展奠定基础。

Objective:To facilitate the use of RNA interference technique in laboratory research, we here made a detailed description of the construction of a siRNA expressing plasmid by PCR method. Methods:The U6+27 promoter cassette and siEGFP sequence were obtained by PCR method. They were cloned into pUC19. The resultant plasmid pU6-siEGFP was co-transfected into COS-7 with pcDNA3.1/EGFP. Green fluorescent expression was observed under fluorescent microscopy. Results:The plasmid pU6-siEGFP can efficiently reduce the expression of EGFP in COS-7 cells. Conclusion:We successfully constructed the siRNA expressing plasmid by PCR method. The construction process is applicable in many laboratories, which facilitates the wider use of RNA interference technique.