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

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 - PLoS ONE (2014)

Bottom Line: While effective, it is not equipped to express multiple siRNAs in a single vector.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.These findings demonstrate that the GDA-based pSOK system has been proven simplistic, effective and versatile for simultaneous expression of multiple siRNAs.

View Article: PubMed Central - PubMed

Affiliation: 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.

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 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.

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Silencing β-catenin diminishes the synergistic osteogenic activity between BMP9 and Wnt3A in iMEF cells.(A) Wnt3A and/or BMP9-induced early osteogenic marker alkaline phosphatase (ALP) activity is reduced in iMEF-simBC3 cells. Subconfluent iMEF-simBC3 and iMEF-siControl cells were infected with AdWnt3A, AdBMP9, AdGFP, or AdWnt3A+AdBMP9. At day 5 post infection, cells were fixed for ALP histochemical staining assay. Each assay conditions were done in triplicate. Representative results are shown. (B) Wnt3A and/or BMP9-induced ALP activity is decreased in the β-catenin silenced iMEFs. The experiments were set up in a similar fashion to that described in (A). At days 3 and 5, cells were lysed for quantitative ALP activity assays. Basal ALP activities (e.g., GFP groups) were subtracted from all BMP9, Wnt3A, and Wnt3A+BMP9 groups. Each assay conditions were done in triplicate. “**”, p<0.001 (iMEF-simBC3 vs. iMEF-siControl).
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pone-0113064-g005: Silencing β-catenin diminishes the synergistic osteogenic activity between BMP9 and Wnt3A in iMEF cells.(A) Wnt3A and/or BMP9-induced early osteogenic marker alkaline phosphatase (ALP) activity is reduced in iMEF-simBC3 cells. Subconfluent iMEF-simBC3 and iMEF-siControl cells were infected with AdWnt3A, AdBMP9, AdGFP, or AdWnt3A+AdBMP9. At day 5 post infection, cells were fixed for ALP histochemical staining assay. Each assay conditions were done in triplicate. Representative results are shown. (B) Wnt3A and/or BMP9-induced ALP activity is decreased in the β-catenin silenced iMEFs. The experiments were set up in a similar fashion to that described in (A). At days 3 and 5, cells were lysed for quantitative ALP activity assays. Basal ALP activities (e.g., GFP groups) were subtracted from all BMP9, Wnt3A, and Wnt3A+BMP9 groups. Each assay conditions were done in triplicate. “**”, p<0.001 (iMEF-simBC3 vs. iMEF-siControl).

Mentions: We further analyzed the functional consequences of β-catenin knockdown on MSC differentiation. We and others demonstrated that canonical Wnt signaling can induce osteogenic differentiation of mesenchymal stem cells [39], [40], [48], [60]. We sought to determine if Wnt3A can induce early osteogenic marker alkaline phosphatase (ALP) activity in iMEFs, and if the induced ALP activity would be reduced when β-catenin is silenced in iMEFs. We found that Wnt3A effectively induced ALP activity in iMEF-siControl cells, which was significantly blunted in iMEF-simBC3 cells (Figure 5A). We previously demonstrated that BMP9 is one of the most potent osteogenic BMPs in mesenchymal cell stems [26], [30], [32], [33], [61]. We found that BMP9 stimulated robust ALP activity in iMEF-siControl cells while the BMP9-induced ALP activity was remarkably reduced in iMEF-simBC3 cells (Figure 5A).


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

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 - PLoS ONE (2014)

Silencing β-catenin diminishes the synergistic osteogenic activity between BMP9 and Wnt3A in iMEF cells.(A) Wnt3A and/or BMP9-induced early osteogenic marker alkaline phosphatase (ALP) activity is reduced in iMEF-simBC3 cells. Subconfluent iMEF-simBC3 and iMEF-siControl cells were infected with AdWnt3A, AdBMP9, AdGFP, or AdWnt3A+AdBMP9. At day 5 post infection, cells were fixed for ALP histochemical staining assay. Each assay conditions were done in triplicate. Representative results are shown. (B) Wnt3A and/or BMP9-induced ALP activity is decreased in the β-catenin silenced iMEFs. The experiments were set up in a similar fashion to that described in (A). At days 3 and 5, cells were lysed for quantitative ALP activity assays. Basal ALP activities (e.g., GFP groups) were subtracted from all BMP9, Wnt3A, and Wnt3A+BMP9 groups. Each assay conditions were done in triplicate. “**”, p<0.001 (iMEF-simBC3 vs. iMEF-siControl).
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getmorefigures.php?uid=PMC4232585&req=5

pone-0113064-g005: Silencing β-catenin diminishes the synergistic osteogenic activity between BMP9 and Wnt3A in iMEF cells.(A) Wnt3A and/or BMP9-induced early osteogenic marker alkaline phosphatase (ALP) activity is reduced in iMEF-simBC3 cells. Subconfluent iMEF-simBC3 and iMEF-siControl cells were infected with AdWnt3A, AdBMP9, AdGFP, or AdWnt3A+AdBMP9. At day 5 post infection, cells were fixed for ALP histochemical staining assay. Each assay conditions were done in triplicate. Representative results are shown. (B) Wnt3A and/or BMP9-induced ALP activity is decreased in the β-catenin silenced iMEFs. The experiments were set up in a similar fashion to that described in (A). At days 3 and 5, cells were lysed for quantitative ALP activity assays. Basal ALP activities (e.g., GFP groups) were subtracted from all BMP9, Wnt3A, and Wnt3A+BMP9 groups. Each assay conditions were done in triplicate. “**”, p<0.001 (iMEF-simBC3 vs. iMEF-siControl).
Mentions: We further analyzed the functional consequences of β-catenin knockdown on MSC differentiation. We and others demonstrated that canonical Wnt signaling can induce osteogenic differentiation of mesenchymal stem cells [39], [40], [48], [60]. We sought to determine if Wnt3A can induce early osteogenic marker alkaline phosphatase (ALP) activity in iMEFs, and if the induced ALP activity would be reduced when β-catenin is silenced in iMEFs. We found that Wnt3A effectively induced ALP activity in iMEF-siControl cells, which was significantly blunted in iMEF-simBC3 cells (Figure 5A). We previously demonstrated that BMP9 is one of the most potent osteogenic BMPs in mesenchymal cell stems [26], [30], [32], [33], [61]. We found that BMP9 stimulated robust ALP activity in iMEF-siControl cells while the BMP9-induced ALP activity was remarkably reduced in iMEF-simBC3 cells (Figure 5A).

Bottom Line: While effective, it is not equipped to express multiple siRNAs in a single vector.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.These findings demonstrate that the GDA-based pSOK system has been proven simplistic, effective and versatile for simultaneous expression of multiple siRNAs.

View Article: PubMed Central - PubMed

Affiliation: 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.

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 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.

Show MeSH
Related in: MedlinePlus