小分子干扰RNA(siRNA)表达载体沉默靶基因的研究

TARGETED GENE SILENCE IN MAMMALIAN CELLS BY USING=VECTORS EXPRESSING SMALL INTERFERING RNA

目的 研究siRNA表达载体在体外培养细胞中对目的基因的干扰作用 ,建立利用RNA干扰技术研究基因功能的平台。 方法 构建针对β actin基因的表达siRNA载体 ,转染HeLa细胞 ,然后在mRNA水平、蛋白水平和细胞形态水平评价siRNA表达载体对 β actin基因表达的抑制作用。 结果 siRNA表达载体可以有效的抑制体外培养细胞 β actin基因的表达 ,使 β actin基因的mRNA和蛋白表达量显著下降 ,细胞形态发生显著变化。 结论siRNA表达载体可以在体外培养细胞中有效的沉默 β actin基因 ,为研究基因功能提供了一个快速、有效的系统。

Objective To study targeted gene silence in mammalian cells by using vector expressing small interfering RNA, in order to establish a platform of gene functions studying using RNAi technology. Methods A plasmid vector expressing βactin siRNAs was constructed, and HeLa cells were transfected using the vector. The expression of βactin gene was assessed in both mRNA and protein levels. Morphological characteristics of the cells were also used to evaluate the function of the vector. Results RTPCR and Western blot results indicated that βactin gene expression was efficiently suppressed by plasmid expressing βactin siRNAs. Morphology of the transfected cells was changed remarkablely. Conclusion The plasmid vector expressing siRNA can induce the silence of βactin gene, and should be an efficient system to study gene functions in mammalian cells.;