哺乳动物细胞siRNA表达载体的构建及鉴定

Construction and identification of a vector expressing siRNA in mammalian cells

目的 :构建哺乳动物细胞siRNA表达载体 .方法 :用PCR的方法扩增人源性的U6启动子的序列 ,并将其克隆入pUC1 8载体中 .进一步将针对绿色荧光蛋白的编码siRNA的DNA克隆入U6启动子的下游 .通过限制性酶切和测序对该重组表达载体进行鉴定 .结果 :限制性酶切和测序结果表明成功构建了哺乳动物细胞siRNA表达载体pUC1 8U6 .结论 :哺乳动物细胞siRNA表达载体pUC1

AIM: To construct a vector expressing siRNA in mammalian cells. METHODS: Using genomic DNA isolated from HepG2 cells as template, human RNA polymerase III U6 promoter was amplified by PCR. The PCR product was then cloned into pUC18 vector. siRNA against green fluorescent protein was cloned into downstream of U6 promoter. The recombinant vector was confirmed by restriction digestion and DNA sequencing. RESULTS: Results of restriction digestion and DNA sequencing showed that the vector for expressing siRNA in mammalian cells was successfully constructed. CONCLUSION: Construction of the siRNA expression vector in mammalian cells lays the foundation for using siRNA to study the function of genes and to treat diseases.