A Simplified and Versatile System for the Simultaneous Expression of Multiple siRNAs in Mammalian Cells Using Gibson DNA Assembly

作者: Fang Deng , Xiang Chen , Zhan Liao , Zhengjian Yan , Zhongliang Wang

DOI: 10.1371/JOURNAL.PONE.0113064

关键词:

摘要: RNA interference (RNAi) denotes sequence-specific mRNA degradation induced by short interfering double-stranded (siRNA) and has become a revolutionary tool for functional annotation of mammalian genes, as well development novel therapeutics. The practical applications RNAi are usually achieved expressing hairpin RNAs (shRNAs) or siRNAs in cells. However, major technical challenge is to simultaneously express multiple silence one more genes. We previously developed pSOS system, which siRNA duplexes made from oligo templates driven opposing U6 H1 promoters. While effective, it not equipped single vector. Gibson DNA Assembly (GDA) an vitro recombination system that the capacity assemble overlapping molecules isothermal step. Here, we GDA-based pSOK assembly constructing vectors sites. fragments were generated PCR amplifications U6-H1 template vector pB2B. GDA specificity was conferred unique sequences insert fragments. To prove feasibility, constructed contain four sites three targeting human mouse β-catenin, respectively. reactions efficient, candidate clones readily identified screening. Multiple β-catenin effectively silenced endogenous expression, inhibited Wnt3A-induced β-catenin/Tcf4 reporter activity expression Wnt/β-catenin downstream Silencing mesenchymal stem cells early osteogenic differentiation significantly diminished synergistic between BMP9 Wnt3A vivo. These findings demonstrate been proven simplistic, effective versatile simultaneous siRNAs. Thus, reported should be valuable gene function studies

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