Selection and validation of optimal siRNA target sites for RNAi-mediated gene silencing

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/TcA 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. (c) 2007 Elsevier B.V. All rights reserved.

RNA干扰（RNAi）介导的基因沉默已成为功能研究、反向基因组学和药物研发的重要工具。使用RNAi的一个主要挑战是确定给定基因最有效的短干扰RNA（siRNA）位点。尽管一些已发表的生物信息学预测模型已被证明是有用的，但为给定基因选择和验证最佳siRNA位点的过程仍然是经验性的且费力的。在此，我们利用逆转录病毒载体骨架（即pSOS）开发了一种基于荧光的筛选系统，其前提是候选siRNA会敲低绿色荧光蛋白（GFP）和靶基因之间的嵌合转录本。siRNA的表达由反向会聚的H1和U6启动子驱动。这种配置简化了双链siRNA寡核苷酸盒的克隆。我们证明了GFP信号的降低与人类β - 连环蛋白的siRNA敲低效率以及β - 连环蛋白/TcA信号活性的抑制密切相关。pSOS不仅应有助于候选siRNA位点的选择和验证，还能通过病毒载体在哺乳动物细胞中提供有效的siRNA传递工具。因此，pSOS系统代表了一种高效且用户友好的选择和验证siRNA靶位点的策略。（c）2007爱思唯尔公司。版权所有。