Journal
ACS SYNTHETIC BIOLOGY
Volume 8, Issue 9, Pages 2092-2105Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.9b00203
Keywords
RNA interference (RNAi); short interfering double-stranded RNA (siRNA); Wnt/beta-catenin signaling; BMP9/Smad4 signaling; mesenchymal stem cells (MSCs); osteogenic differentiation; multiplex expression; shotgun cloning
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Funding
- National Key Research and Development Program of China [2016YFC1000803, 2011CB707906]
- National Institutes of Health [CA226303]
- U.S. Department of Defense [OR130096]
- Scoliosis Research Society
- University of Chicago Cancer Center Support Grant [P30CA014599]
- National Center for Advancing Translational Sciences of the National Institutes of Health [UL1 TR000430]
- Mabel Green Myers Research Endowment Fund
- University of Chicago Orthopaedics Alumni Fund
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As an important post-transcriptional regulatory machinery mediated by similar to 21nt short-interfering double-stranded RNA (siRNA), RNA interference (RNAi) is a powerful tool to delineate gene functions and develop therapeutics. However, effective RNAi-mediated silencing requires multiple siRNAs for given genes, a time-consuming process to accomplish. Here, we developed a user-friendly system for single-vector-based multiplex siRNA expression by exploiting the unique feature of restriction endonuclease BstXI. Specifically, we engineered a BstXI-based shotgun cloning (BSG) system, which consists of three entry vectors with siRNA expression units (SiEUs) flanked with distinct BstXI sites, and a retroviral destination vector for shotgun SiEU assembly. For proof-of-principle studies, we constructed multiplex siRNA vectors silencing beta-catenin and/or Smad4 and assessed their functionalities in mesenchymal stem cells (MSCs). Pooled siRNA cassettes were effectively inserted into respective entry vectors in one-step, and shotgun seamless assembly of pooled BstXI-digested SiEU fragments into a retroviral destination vector followed. We found these multiplex siRNAs effectively silenced beta-catenin and/or Smad4, and inhibited Wnt3A- or BMP9-specific reporters and downstream target expression in MSCs. Furthermore, multiplex silencing of beta-catenin and/or Smad4 diminished Wnt3A and/or BMP9-induced osteogenic differentiation. Collectively, the BSG system is a user-friendly technology for single-vector-based multiplex siRNA expression to study gene functions and develop experimental therapeutics.
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