A simplified and versatile system for the simultaneous expression of multiple siRNAs in mammalian cells using Gibson DNA Assembly.
A simplified and versatile system for the simultaneous expression of multiple siRNAs in mammalian cells using Gibson DNA Assembly.
复制标题
DOI：
10.1371/journal.pone.0113064
复制
发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
He TC
中科院分区：
文献类型：
--
作者：
Deng F;Chen X;Liao Z;Yan Z;Wang Z;Deng Y;Zhang Q;Zhang Z;Ye J;Qiao M;Li R;Denduluri S;Wang J;Wei Q;Li M;Geng N;Zhao L;Zhou G;Zhang P;Luu HH;Haydon RC;Reid RR;Yang T;He TC
RNA干扰（RNA interference，RNAi）是指由短的干扰双链RNA（siRNA）诱导的序列特异性mRNA降解，已成为哺乳动物基因功能注释和开发新型治疗药物的革命性工具。RNAi的实际应用通常通过在细胞中表达短发夹RNA（shRNAs）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。我们先前开发了pSOS系统，其中siRNA双链体由相对的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，其具有在单个等温步骤中组装多个重叠DNA分子的能力。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。通过PCR扩增从U6-H1模板载体pB 2B产生组装片段。GDA组装特异性由插入片段的重叠的独特siRNA序列赋予。为了证明技术上的可行性，我们构建了分别含有针对人和小鼠β-catenin的四个siRNA位点和三个siRNA位点的pSOK载体。组装反应是有效的，并且候选克隆容易通过PCR筛选鉴定。多个β-catenin siRNA有效地沉默内源性β-catenin表达，抑制Wnt 3A诱导的β-catenin/Tcf 4报告基因活性和Wnt/β-catenin下游基因的表达。沉默间充质干细胞中的β-catenin抑制Wnt 3A诱导的早期成骨分化，并在体外和体内显著降低BMP 9和Wnt 3A之间的协同成骨活性。这些发现表明，基于GDA的pSOK系统已被证明是简单的，有效的和通用的多个siRNA的同时表达。因此，报告的pSOK系统应该是一个有价值的工具，基因功能的研究和开发新的治疗方法。
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.
登录
查看更多内容
影响因子：
21.3
作者：
通讯作者：
--
条件永生化小鼠胚胎成纤维细胞保留增殖活性而不影响多能分化潜能
影响因子：
3.7
作者：
Huang E;Bi Y;Jiang W;Luo X;Yang K;Gao JL;Gao Y;Luo Q;Shi Q;Kim SH;Liu X;Li M;Hu N;Liu H;Cui J;Zhang W;Li R;Chen X;Shen J;Kong Y;Zhang J;Wang J;Luo J;He BC;Wang H;Reid RR;Luu HH;Haydon RC;Yang L;He TC
通讯作者：
He TC
DOI：
10.1038/nrg3355
发表时间：
2013-02
期刊：
Nature reviews. Genetics
影响因子：
--
作者：
通讯作者：
--
生长激素通过激活小鼠多系细胞中的 JAK/STAT/IGF1 通路与 BMP9 协同促进成骨分化
影响因子：
6.2
作者：
Huang, Enyi;Zhu, Gaohui;He, Tong-Chuan
通讯作者：
He, Tong-Chuan
影响因子：
48
作者：
Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
通讯作者：
Smith, Hamilton O.