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A simplified and versatile system for the simultaneous expression of multiple siRNAs in mammalian cells using Gibson DNA Assembly.

ABSTRACT: 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 ?-catenin, respectively. The assembly reactions were efficient, and candidate clones were readily identified by PCR screening. Multiple ?-catenin siRNAs effectively silenced endogenous ?-catenin expression, inhibited Wnt3A-induced ?-catenin/Tcf4 reporter activity and expression of Wnt/?-catenin downstream genes. Silencing ?-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.

SUBMITTER: Deng F

PROVIDER: S-EPMC4232585 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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A simplified and versatile system for the simultaneous expression of multiple siRNAs in mammalian cells using Gibson DNA Assembly.

Deng Fang F Chen Xiang X Liao Zhan Z Yan Zhengjian Z Wang Zhongliang Z Deng Youlin Y Zhang Qian Q Zhang Zhonglin Z Ye Jixing J Qiao Min M Li Ruifang R Denduluri Sahitya S Wang Jing J Wei Qiang Q Li Melissa M Geng Nisha N Zhao Lianggong L Zhou Guolin G Zhang Penghui P Luu Hue H HH Haydon Rex C RC Reid Russell R RR Yang Tian T He Tong-Chuan TC

PloS one 20141114 11

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 ...[more]

PMID: 25398142

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Project description:BackgroundSite-directed mutagenesis (SDM) is a key method in molecular biology; allowing to modify DNA sequences at single base pair resolution. Although many SDM methods have been developed, methods that increase efficiency and versatility of this process remain highly desired.MethodWe present a versatile and simple method to efficiently introduce a variety of mutation schemes using Gibson-assembly but without the need to design uniquely designated Gibson primers. Instead, we explore the re-use of standard SDM primers (completely overlapping in sequence) in combination with regular primers (~ 25 bps long) for amplification of fragments flanking the site of mutagenesis. We further introduce a rapid amplification step of the Gibson-assembled product for analysis and quality control, as well as for ligation, or re-ligation at instances the process fails (avoiding expenditure of added Gibson reaction mixtures).ResultsWe first demonstrate that standard SDM primers can be used with the Gibson assembly method and, despite the need for extensive digestion of the DNA past the entire primer sequence, the reaction is attainable within as short as 15 min. We also find that the amount of the assembled Gibson product is too low to be visualized on standard agarose gel. Our added amplification step (by use of the same short primers initially employed) remedies this limitation and allows to resolve whether the desired Gibson-assembled product has been obtained on agarose gel or by sequencing of amplicons. It also provides large amounts of amplicons for subsequent ligations, bypassing the need to re-employ Gibson mixtures. Lastly, we find that our method can easily accommodate SDM primers with degenerate sequences.ConclusionWe employ our alternative approach to delete, replace, insert, and degenerate sequences within target DNA sequences, specifically DNA sequences that proved very resistant to mutagenesis by multiple other SDM methods (standard and commercial). Importantly, our approach involves the re-use of SDM primers from our primer-inventory. Our scheme thereby reduces the need (and time and money) to design and order new custom Gibson-primers. Together, we provide a simple and versatile protocol that spans only 4 days (including the added amplification step), requires minimal primer sets and provides very high yields and success rates (> 98%).

Dataset Information

A simplified and versatile system for the simultaneous expression of multiple siRNAs in mammalian cells using Gibson DNA Assembly.

Publications

A simplified and versatile system for the simultaneous expression of multiple siRNAs in mammalian cells using Gibson DNA Assembly.

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