应用RNA干扰抑制目的基因在小鼠体内表达

目的研究RNA干扰(RNAi)对体外和体内基因表达的沉默作用。方法以PCR方法从基因组DNA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;构建特异性针对目的基因(绿色荧光蛋白,GFP)的RNAi载体(Pi)。将RNAi干扰载体和GFP载体转染NIH3T3细胞,应用荧光显微镜观察、RT-PCR、流式细胞技术(FACS)分析上述细胞中RNAi对目的基因GFP表达的抑制效果,进一步在个体水平将RNAi载体注入小鼠体内,研究RNAi对GFP表达的抑制情况。结果RNAi能有效地使NIH3T3细胞中GFP表达量降低约60%以上,并能有效地抑制GFP在转基因小鼠体内的表达。结论RNAi技术能抑制目的基因在体内外的表达,是一种有效的基因治疗工具。

Suppression of gene expression in cells by RNA interference

[Objective] To evaluate whether RNA interference (RNAi) can silence gene expression in vitro and in vivo. [Methods] H1 promoter was amplified from human genome DNA for driving RNAi synthesis by PCR. RNAi vectors (Pi) for silencing the GFP expression were constructed. The GFP vector and RNAi vector (Pi) were then co-transfected into the NIH3T3, in which the silencing effect on GFP expression was investigated by fluorescence microscopic observation, RT-PCR and FACS analysis. Then RNAi plasmid was injected into GFP transgenic mice by tail vein to investigate the silencing effect of RNAi to GFP expression in vivo. [Results] RNAi could effectively reduce more than 60 percent of GFP expression in NIH3T3 cells as well as inhibit the GFP expression in GFP transgenic mice. [Conclusions] RNAi technology, which can inhibit gene expression in cells, is an effective tool for gene therapy.