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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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Multiple siRNAs targeting mouse β-catenin simBC3 effectively inhibit canonical Wnt signaling activity in iMEFs.(A) Reduced β-catenin expression in iMEF-simBC3 cells. Subconfluent iMEF-simBC3 and iMEF-siControl cells were infected with AdWnt3A or AdGFP. At 36 h after infection, total RNA was isolated and subjected to qPCR analysis using primers for mouse β-catenin and GAPDH. Relative expression was calculated by dividing the β-catenin expression levels with respective GAPDH expression. All samples were subjected to the subtraction of baseline (i.e., AdGFP infected cells) expression. Each assay was done in triplicate. “**”, p<0.001. (B) iMEF-simBC3 cells exhibit significantly lower β-catenin/Tcf reporter activity upon Wnt3A stimulation. Subconfluent iMEF-simBC3 and iMEF-siControl cells were transfected with TOP-Luc reporter plasmid and infected with AdWnt3A or AdGFP. At 24 h and 48 h post transfection/infection, cells were lysed for luciferase assays. Relative β-catenin/Tcf reporter activity was subjected to subtractions of basal activity (i.e., AdGFP groups). Easy conditions were done in triplicate. “**”, p<0.001. (C). Wnt3A-induced expression of Wnt/β-catenin target genes was significantly decreased in iMEF-simBC3 cells. Subconfluent iMEF-simBC3 and iMEF-siControl cells were infected with AdWnt3A or AdGFP for 36 h. Total RNA was isolated and subjected to reverse transcription. The resultant cDNAs were used as templates for qPCR analysis using primers specific for mouse Axin2 and c-Myc transcripts. All samples were normalized by GAPDH levels. Each assay condition was done in triplicate. “**”, p<0.001. (D) simBC3 can effectively block Wnt3a-induced β-catenin accumulation. Subconfluent iMEF-siControl (a) cells fixed and subjected to immunofluorescence staining with an anti-β-catenin antibody. The cell nuclei were counter stained with DAPI. Control IgG and minus primary antibody were used as negative controls (data not shown). Representative images are shown.
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pone-0113064-g004: Multiple siRNAs targeting mouse β-catenin simBC3 effectively inhibit canonical Wnt signaling activity in iMEFs.(A) Reduced β-catenin expression in iMEF-simBC3 cells. Subconfluent iMEF-simBC3 and iMEF-siControl cells were infected with AdWnt3A or AdGFP. At 36 h after infection, total RNA was isolated and subjected to qPCR analysis using primers for mouse β-catenin and GAPDH. Relative expression was calculated by dividing the β-catenin expression levels with respective GAPDH expression. All samples were subjected to the subtraction of baseline (i.e., AdGFP infected cells) expression. Each assay was done in triplicate. “**”, p<0.001. (B) iMEF-simBC3 cells exhibit significantly lower β-catenin/Tcf reporter activity upon Wnt3A stimulation. Subconfluent iMEF-simBC3 and iMEF-siControl cells were transfected with TOP-Luc reporter plasmid and infected with AdWnt3A or AdGFP. At 24 h and 48 h post transfection/infection, cells were lysed for luciferase assays. Relative β-catenin/Tcf reporter activity was subjected to subtractions of basal activity (i.e., AdGFP groups). Easy conditions were done in triplicate. “**”, p<0.001. (C). Wnt3A-induced expression of Wnt/β-catenin target genes was significantly decreased in iMEF-simBC3 cells. Subconfluent iMEF-simBC3 and iMEF-siControl cells were infected with AdWnt3A or AdGFP for 36 h. Total RNA was isolated and subjected to reverse transcription. The resultant cDNAs were used as templates for qPCR analysis using primers specific for mouse Axin2 and c-Myc transcripts. All samples were normalized by GAPDH levels. Each assay condition was done in triplicate. “**”, p<0.001. (D) simBC3 can effectively block Wnt3a-induced β-catenin accumulation. Subconfluent iMEF-siControl (a) cells fixed and subjected to immunofluorescence staining with an anti-β-catenin antibody. The cell nuclei were counter stained with DAPI. Control IgG and minus primary antibody were used as negative controls (data not shown). Representative images are shown.

Mentions: Our results in Figure 2 indicate that three siRNA sites (two inserts) are seemingly more favorably assembled into pSOK vector. It is conceivable that in most cases three siRNA sites should be sufficiently effective in silencing a given gene. Here, we tested this possibility by constructing a vector, designated as pSOK-simBC, that expressed three siRNA sites targeting mouse β-catenin (Figure 1B, panel b). The construction and screening process were very efficient. After sequencing verification, the pSOK-simBC was packaged as retrovirus and used to generate the stable line iMEF-simBC3, along with a control line iMEF-siControl. The iMEFs were previously characterized multi-potent mesenchymal stem cells (MSCs) [20], [25]. When the iMEF stable lines were infected with AdWnt3A or AdGFP and analyzed for β-catenin expression, we found that β-catenin expression was significantly reduced in iMEF-simBC3 cells, compared with that in iMEF-siControl cells (p<0.001) (Figure 4A).


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)

Multiple siRNAs targeting mouse β-catenin simBC3 effectively inhibit canonical Wnt signaling activity in iMEFs.(A) Reduced β-catenin expression in iMEF-simBC3 cells. Subconfluent iMEF-simBC3 and iMEF-siControl cells were infected with AdWnt3A or AdGFP. At 36 h after infection, total RNA was isolated and subjected to qPCR analysis using primers for mouse β-catenin and GAPDH. Relative expression was calculated by dividing the β-catenin expression levels with respective GAPDH expression. All samples were subjected to the subtraction of baseline (i.e., AdGFP infected cells) expression. Each assay was done in triplicate. “**”, p<0.001. (B) iMEF-simBC3 cells exhibit significantly lower β-catenin/Tcf reporter activity upon Wnt3A stimulation. Subconfluent iMEF-simBC3 and iMEF-siControl cells were transfected with TOP-Luc reporter plasmid and infected with AdWnt3A or AdGFP. At 24 h and 48 h post transfection/infection, cells were lysed for luciferase assays. Relative β-catenin/Tcf reporter activity was subjected to subtractions of basal activity (i.e., AdGFP groups). Easy conditions were done in triplicate. “**”, p<0.001. (C). Wnt3A-induced expression of Wnt/β-catenin target genes was significantly decreased in iMEF-simBC3 cells. Subconfluent iMEF-simBC3 and iMEF-siControl cells were infected with AdWnt3A or AdGFP for 36 h. Total RNA was isolated and subjected to reverse transcription. The resultant cDNAs were used as templates for qPCR analysis using primers specific for mouse Axin2 and c-Myc transcripts. All samples were normalized by GAPDH levels. Each assay condition was done in triplicate. “**”, p<0.001. (D) simBC3 can effectively block Wnt3a-induced β-catenin accumulation. Subconfluent iMEF-siControl (a) cells fixed and subjected to immunofluorescence staining with an anti-β-catenin antibody. The cell nuclei were counter stained with DAPI. Control IgG and minus primary antibody were used as negative controls (data not shown). Representative images are shown.
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pone-0113064-g004: Multiple siRNAs targeting mouse β-catenin simBC3 effectively inhibit canonical Wnt signaling activity in iMEFs.(A) Reduced β-catenin expression in iMEF-simBC3 cells. Subconfluent iMEF-simBC3 and iMEF-siControl cells were infected with AdWnt3A or AdGFP. At 36 h after infection, total RNA was isolated and subjected to qPCR analysis using primers for mouse β-catenin and GAPDH. Relative expression was calculated by dividing the β-catenin expression levels with respective GAPDH expression. All samples were subjected to the subtraction of baseline (i.e., AdGFP infected cells) expression. Each assay was done in triplicate. “**”, p<0.001. (B) iMEF-simBC3 cells exhibit significantly lower β-catenin/Tcf reporter activity upon Wnt3A stimulation. Subconfluent iMEF-simBC3 and iMEF-siControl cells were transfected with TOP-Luc reporter plasmid and infected with AdWnt3A or AdGFP. At 24 h and 48 h post transfection/infection, cells were lysed for luciferase assays. Relative β-catenin/Tcf reporter activity was subjected to subtractions of basal activity (i.e., AdGFP groups). Easy conditions were done in triplicate. “**”, p<0.001. (C). Wnt3A-induced expression of Wnt/β-catenin target genes was significantly decreased in iMEF-simBC3 cells. Subconfluent iMEF-simBC3 and iMEF-siControl cells were infected with AdWnt3A or AdGFP for 36 h. Total RNA was isolated and subjected to reverse transcription. The resultant cDNAs were used as templates for qPCR analysis using primers specific for mouse Axin2 and c-Myc transcripts. All samples were normalized by GAPDH levels. Each assay condition was done in triplicate. “**”, p<0.001. (D) simBC3 can effectively block Wnt3a-induced β-catenin accumulation. Subconfluent iMEF-siControl (a) cells fixed and subjected to immunofluorescence staining with an anti-β-catenin antibody. The cell nuclei were counter stained with DAPI. Control IgG and minus primary antibody were used as negative controls (data not shown). Representative images are shown.
Mentions: Our results in Figure 2 indicate that three siRNA sites (two inserts) are seemingly more favorably assembled into pSOK vector. It is conceivable that in most cases three siRNA sites should be sufficiently effective in silencing a given gene. Here, we tested this possibility by constructing a vector, designated as pSOK-simBC, that expressed three siRNA sites targeting mouse β-catenin (Figure 1B, panel b). The construction and screening process were very efficient. After sequencing verification, the pSOK-simBC was packaged as retrovirus and used to generate the stable line iMEF-simBC3, along with a control line iMEF-siControl. The iMEFs were previously characterized multi-potent mesenchymal stem cells (MSCs) [20], [25]. When the iMEF stable lines were infected with AdWnt3A or AdGFP and analyzed for β-catenin expression, we found that β-catenin expression was significantly reduced in iMEF-simBC3 cells, compared with that in iMEF-siControl cells (p<0.001) (Figure 4A).

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