利用人U6 snRNA启动子构建RNA干扰质粒载体

RNA干扰技术已经成为基因功能研究等领域的有力工具，构建带有筛选标记的siRNA载体可以在细胞中持续抑制靶基因的表达.为了利用RNAi技术开展生物学研究，在克隆载体pUC19的基础上改造构建了人类细胞小干扰RNA（small interference RNA，siRNA）表达质粒pUC19NU.该质粒具有新霉素抗性标记和真核细胞复制起点，利用连入的人U6 snRNA启动子起始siRNA的转录.以EGFP 和p53为靶基因的干扰实验证明，所构建的siRNA表达质粒可以显著抑制细胞外源性增强绿色荧光蛋白（enhanced green fluorescent protein，EGFP）及细胞内源性p53蛋白的表达，而且抑制效果具有特异性.

Constructing of an RNAi Plasmid Based on Human U6 snRNA

RNA interference is widely used in the field of gene function research. A siRNA vector with selective tag can retain the interfering effect on the target gene expression. In this study, an RNAi plasmid containing the U6 promoter was constructed based on the backbone of pUC19. This vector has the character of neomycin-resistant and the ability of replicating in eukaryotic cells.Two genes were selected as the interfering target: the enhanced green fluorescent protein(EGFP) as a exogenously gene and the p53 protein as a endogenously gene. Fluorescent microscope image showed that over 72 hours period following transient transfecting vector pUC19NUE into HeLa cell line,either the number of the cells emitting green fluorescent or the fluorescent intensity were sustained lower compared with the control.While transfecting vector pUC19NUP harboring RNAi for p53 mRNA led to time-dependent reduction of p53 proteins showed by Western blot,being decline at 72 hours;flow cytometry assay further demonstrated that these cells with down-regulation of p53 gene expression were delayed for entering S phase of cell cycle.These results suggested that U6 promoter-driven siRNA efficiently suppress targeted gene expression in mammalian cells.