| Summary: | 碩士 === 國立成功大學 === 生物化學研究所 === 93 === Small interfering RNAs (siRNA) have become the most powerful gene silencing reagents for studying gene functions in mammalian systems. However, some experimental evidences have already shown that only a limited number of siRNAs can induce highly effective RNA interference (RNAi) in mammalian cells. Moreover, the efficacy of siRNAs can only be experimentally measured based on suppression of the target gene expression. To achieve the functional screening for effective siRNAs more robustly and cost-effectively, we have developed a reliable and quantitative reporter-based siRNA validation system that eliminated the need for cDNA clones. This system consisted of a short synthetic DNA fragment including an RNAi targeting sequence of interest and two expression vectors for targeting reporter as well as triggering siRNA expression. In order to generate the targeting reporter vector for this system, the short synthetic DNA fragment, instead of cDNA, was fused with a reporter gene at the 5’-or 3’-untranslated region (5’-or 3’-UTR) or inserted within the reporter gene without interrupting its activity. Simultaneously, this DNA fragment was also cloned into the triggering siRNA expression vector, pDual, which contained two convergent RNA polymerase III (Pol III) promoters, mouse U6 and human H1, to drive expression of the sense and antisense strands of siRNA, respectively. The efficacy of the siRNAs is determined by their abilities to inhibit expression of the targeting reporter gene with easily quantified readouts including enhanced green fluorescence protein (EGFP) or firefly luciferase. By using fully analyzed siRNAs directed against human hepatitis B virus (HBV) surface antigen (HBsAg) or tumor suppressor protein p53 (Trp53) gene expression, we have demonstrated that this reporter-based siRNA validation system could effectively and faithfully report the efficacy of the corresponding siRNAs in a sequence-specific manner. In addition, we have further identified the potent siRNAs for silencing mouse MMP-7 gene expression by using this system. Furthermore, H1 promoter is often used to express siRNA or shRNA, and it has a bidirectional character. We have confirmed H1 promoter was a bidirectional promoter by using EGFP as well as firefly luciferase genes. In addition, we have selected stable cell lines expressing Bsd and shLuc by applying H1 promoter.
|
|---|
