应用RNA干扰技术调节大鼠c-jun基因的表达

目的:应用RNA干扰(RNAi)技术调节大鼠c-jun基因在Cos-7细胞中的表达.方法:分别构建大鼠c-jun基因的RNA干扰载体和真核表达GFP(绿色荧光蛋白)载体,将两者共转染Cos-7细胞,镜下观察大鼠C-Jun-GFP融合蛋白的表达,应用Western blot方法检测抑制效率.结果:酶切和测序结果表明,大鼠c-jun基因的RNA干扰载体和真核表达GFP载体构建成功,镜下及Western blot共转染结果均显示随着RNA干扰载体浓度的增加C-Jun-GFP融合蛋白表达量逐渐减少.结论:在Cos-7细胞中应用RNAi技术成功调节大鼠c-jun基因的表达.

Using RNA Interference Technique to Regulate the Expression of Rat c-jun Gene

Objective:To regulate the expression of rat c-jun gene in Cos-7 cells by using RNA interference(RNAi)technique to further clarify the relationship between c-jun and diseases.Methods:The siRNA(small interference RNA)expression vector of rat c-jun gene and the eukaryon expression vector were constructed.The two plasmids were transfected into Cos-7 cells together by Lipofec- tamine2000.The inhibitive effect was judged through observing the expression of GFP(greeh fluorescent protein)in Cos-7 cells under inversion fluorescence microscopy after 24h,and then detected it by Western blot.Results:Identifcation by enzyme cutting,PCR and se- quencing showed that siRNA expression vector of rat c-jun gene and the eukaryon expression vector were successfully constructed.After transfecting,the results of inversion fluorescence microscopy and Western blot showed that C-Jun-GFP expressed in Cos-7 cells success- fully.But its expression decreased obviously after cotransfection,and it was decreasing gradually with inereasi(?)of RNAi vector.Con- clusions:The expression of rat c-jun gene in Cos-7 cells can be inhibited significantly by using plasmid-based RNAi.