Deng Fang, Chen Xiang, Liao Zhan, Yan Zhengjian, Wang Zhongliang, Deng Youlin, Zhang Qian, Zhang Zhonglin, Ye Jixing, Qiao Min, Li Ruifang, Denduluri Sahitya, Wang Jing, Wei Qiang, Li Melissa, Geng Nisha, Zhao Lianggong, Zhou Guolin, Zhang Penghui, Luu Hue H, Haydon Rex C, Reid Russell R, Yang Tian, He Tong-Chuan
Department of Cell Biology, Third Military Medical University, Chongqing, 400038, China; Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, 60637, United States of America.
Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, 60637, United States of America.
PLoS One. 2014 Nov 14;9(11):e113064. doi: 10.1371/journal.pone.0113064. eCollection 2014.
RNA interference (RNAi) denotes sequence-specific mRNA degradation induced by short interfering double-stranded RNA (siRNA) and has become a revolutionary tool for functional annotation of mammalian genes, as well as for development of novel therapeutics. The practical applications of RNAi are usually achieved by expressing short hairpin RNAs (shRNAs) or siRNAs in cells. However, a major technical challenge is to simultaneously express multiple siRNAs to silence one or more genes. We previously developed pSOS system, in which siRNA duplexes are made from oligo templates driven by opposing U6 and H1 promoters. While effective, it is not equipped to express multiple siRNAs in a single vector. Gibson DNA Assembly (GDA) is an in vitro recombination system that has the capacity to assemble multiple overlapping DNA molecules in a single isothermal step. Here, we developed a GDA-based pSOK assembly system for constructing single vectors that express multiple siRNA sites. The assembly fragments were generated by PCR amplifications from the U6-H1 template vector pB2B. GDA assembly specificity was conferred by the overlapping unique siRNA sequences of insert fragments. To prove the technical feasibility, we constructed pSOK vectors that contain four siRNA sites and three siRNA sites targeting human and mouse β-catenin, respectively. The assembly reactions were efficient, and candidate clones were readily identified by PCR screening. Multiple β-catenin siRNAs effectively silenced endogenous β-catenin expression, inhibited Wnt3A-induced β-catenin/Tcf4 reporter activity and expression of Wnt/β-catenin downstream genes. Silencing β-catenin in mesenchymal stem cells inhibited Wnt3A-induced early osteogenic differentiation and significantly diminished synergistic osteogenic activity between BMP9 and Wnt3A in vitro and in vivo. These findings demonstrate that the GDA-based pSOK system has been proven simplistic, effective and versatile for simultaneous expression of multiple siRNAs. Thus, the reported pSOK system should be a valuable tool for gene function studies and development of novel therapeutics.
RNA干扰（RNAi）是指由短干扰双链RNA（siRNA）诱导的序列特异性mRNA降解，它已成为用于哺乳动物基因功能注释以及新型治疗药物开发的革命性工具。RNAi的实际应用通常通过在细胞中表达短发夹RNA（shRNA）或siRNA来实现。然而，一个主要的技术挑战是同时表达多个siRNA以沉默一个或多个基因。我们之前开发了pSOS系统，其中siRNA双链体由由相反的U6和H1启动子驱动的寡核苷酸模板制成。虽然有效，但它不具备在单个载体中表达多个siRNA的能力。吉布森DNA组装（GDA）是一种体外重组系统，能够在单个等温步骤中组装多个重叠的DNA分子。在这里，我们开发了一种基于GDA的pSOK组装系统，用于构建表达多个siRNA位点的单个载体。组装片段通过从U6-H1模板载体pB2B进行PCR扩增产生。GDA组装特异性由插入片段的重叠独特siRNA序列赋予。为了证明技术可行性，我们构建了分别包含四个siRNA位点和三个靶向人及小鼠β-连环蛋白的siRNA位点的pSOK载体。组装反应高效，通过PCR筛选很容易鉴定出候选克隆。多个β-连环蛋白siRNA有效沉默内源性β-连环蛋白表达，抑制Wnt3A诱导的β-连环蛋白/Tcf4报告基因活性以及Wnt/β-连环蛋白下游基因的表达。在间充质干细胞中沉默β-连环蛋白抑制Wnt3A诱导的早期成骨分化，并在体外和体内显著降低BMP9和Wnt3A之间的协同成骨活性。这些发现表明，基于GDA的pSOK系统已被证明对于同时表达多个siRNA简单、有效且通用。因此，报道的pSOK系统应该是基因功能研究和新型治疗药物开发的有价值工具。
