Developing a Versatile Shotgun Cloning Strategy for Single-Vector-Based Multiplex Expression of Short Interfering RNAs (siRNAs) in Mammalian Cells

ACS Synth Biol. 2019 Sep 20;8(9):2092-2105. doi: 10.1021/acssynbio.9b00203. Epub 2019 Sep 10.


As an important post-transcriptional regulatory machinery mediated by ∼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 β-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 β-catenin and/or Smad4, and inhibited Wnt3A- or BMP9-specific reporters and downstream target expression in MSCs. Furthermore, multiplex silencing of β-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.

Keywords: BMP9/Smad4 signaling; RNA interference (RNAi); Wnt/β-catenin signaling; mesenchymal stem cells (MSCs); multiplex expression; osteogenic differentiation; short interfering double-stranded RNA (siRNA); shotgun cloning.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Cell Differentiation
  • Cell Line
  • Cloning, Molecular
  • Genetic Vectors / genetics
  • Genetic Vectors / metabolism
  • Growth Differentiation Factor 2 / antagonists & inhibitors
  • Growth Differentiation Factor 2 / genetics
  • Growth Differentiation Factor 2 / metabolism
  • Mesenchymal Stem Cells / cytology
  • Mesenchymal Stem Cells / metabolism
  • Mice
  • Osteogenesis
  • RNA Interference*
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism*
  • Smad4 Protein / antagonists & inhibitors
  • Smad4 Protein / genetics
  • Smad4 Protein / metabolism
  • Wnt Signaling Pathway / genetics
  • beta Catenin / antagonists & inhibitors
  • beta Catenin / genetics
  • beta Catenin / metabolism


  • Growth Differentiation Factor 2
  • RNA, Small Interfering
  • Smad4 Protein
  • beta Catenin