Selection and Validation of Optimal Sirna Target Sites for Rnai-mediated Gene Silencing.
From: The Children’s Hospital, and the Key Laboratory of Diagnostic Medicine designated by the Ministry of Education, Chongqing University of Medical Sciences, Chongqing 400016, China.
Gene
- Publish Date: Jun 2007
- ISSN: 0378-1119
- Volume: 395
- Issue: 1-2
- Pages: 160-9
- Medium: Print
- Language: English
- Citation (JAMA): Luo Qing, Kang Quan, Song Wen-Xin, et al. Selection and Validation of Optimal Sirna Target Sites for Rnai-mediated Gene Silencing.. Gene Jun 2007;395:160-9
Abstract
RNA interference (RNAi)-mediated gene silencing has become a valuable tool for functional studies, reverse genomics, and drug discoveries. One major challenge of using RNAi is to identify the most effective short interfering RNAs (siRNAs) sites of a given gene. Although several published bioinformatic prediction models have proven useful, the process to select and validate optimal siRNA sites for a given gene remains empirical and laborious. Here, we developed a fluorescence-based selection system using a retroviral vector backbone, namely pSOS, which was based on the premise that candidate siRNAs would knockdown the chimeric transcript between GFP and target gene. The expression of siRNA was driven by the opposing convergent H1 and U6 promoters. This configuration simplifies the cloning of duplex siRNA oligonucleotide cassettes. We demonstrated that GFP signal reduction was closely correlated with siRNA knockdown efficiency of human beta-catenin, as well as with the inhibition of beta-catenin/Tcf4 signaling activity. The pSOS should not only facilitate the selection and validation of candidate siRNA sites, but also provide efficient delivery tools of siRNAs via viral vectors in mammalian cells. Thus, the pSOS system represents an efficient and user-friendly strategy to select and validate siRNA target sites.
Mesh Headings (Keywords): Base Sequence, Binding Sites, Cell Line, DNA, Gene Expression, Genes, Reporter, Genetic Techniques, Genetic Vectors, Green Fluorescent Proteins, Humans, Promoter Regions (Genetics), RNA Interference, RNA, Small Interfering, Recombinant Proteins, Transfection, beta Catenin
Check for Full Text / PubMed Unique Identifier (PMID): 17449199
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