应用RNAi抑制细胞中绿色荧光蛋白的表达

Suppression of enhanced green fluorescent protein expression in cells by RNA interference

目的在293T和Mel细胞中研究RNA干扰(RNAinterference,RNAi)对绿色荧光蛋白(enhancedgreenfluorescentprotein,eGFP)表达的沉默作用。方法以巢式PCR方法从人胚肾293T细胞中克隆出依赖于RNA聚合酶Ⅲ的H1启动子,并用于驱动RNAi片段的合成;构建能抑制eGFP特异性表达的RNAi载体(TR1)。将eGFP载体和RNAi干扰载体共转染293T和Mel两类细胞中,应用荧光显微镜观察、逆转录PCR、荧光辅助细胞分选技术和定量逆转录PCR方法分析上述细胞中RNAi对eGFP表达的抑制情况。结果RNAi载体能有效地使293T和Mel细胞中红系特异的eGFP表达量均降低约50%以上。结论本文构建的RNAi载体能有效的抑制目的基因eGFP在细胞中的表达。

Objective To evaluate the role of RNA interference （RNAi） in silencing the enhanced green fluorescent protein （eGFP） expression in 293T and Mel cells. Methods Nested-PCR was used to amplify H1 promoter from human 293T cells for driving RNAi synthesis. RNAi vectors （TR1） for silencing the eGFP expression was constructed. The eGFP veetor and RNAi vector （TR1） were then co-transfected into the 293T and Mel cells, in which the silencing effect on eGFP expression was investigated by fluorescence microscopy, reverse transcription-PCR（RT-PCR）, fluorescence-assited cell sorting（FACS） analysis and real-time RT-PCR. Results RNAi could effectively reduce more than 50 percent of eGFP expression in 293T cells as well as in Mel cells. Conclusion The RNAi vector constructed in this way paper can effectively inhibit eGFP expression in cells.