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Developing a Versatile Shotgun Cloning Strategy for Single-Vector-Based Multiplex Expression of Short Interfering RNAs (siRNAs) in Mammalian Cells
- Source :
- ACS Synthetic Biology
- Publication Year :
- 2019
-
Abstract
- 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.
- Subjects :
- 0106 biological sciences
RNA interference (RNAi)
Small interfering RNA
BMP9/Smad4 signaling
Genetic Vectors
Biomedical Engineering
Wnt β catenin signaling
osteogenic differentiation
multiplex expression
Biology
01 natural sciences
Biochemistry, Genetics and Molecular Biology (miscellaneous)
Cell Line
03 medical and health sciences
Mice
RNA interference
Osteogenesis
010608 biotechnology
Growth Differentiation Factor 2
Animals
Multiplex
Vector (molecular biology)
Cloning, Molecular
RNA, Small Interfering
Gene
Wnt Signaling Pathway
beta Catenin
030304 developmental biology
Smad4 Protein
0303 health sciences
mesenchymal stem cells (MSCs)
Shotgun sequencing
RNA
Cell Differentiation
Mesenchymal Stem Cells
General Medicine
Cell biology
short interfering double-stranded RNA (siRNA)
shotgun cloning
RNA Interference
Research Article
Wnt/β-catenin signaling
Subjects
Details
- ISSN :
- 21615063
- Volume :
- 8
- Issue :
- 9
- Database :
- OpenAIRE
- Journal :
- ACS synthetic biology
- Accession number :
- edsair.doi.dedup.....5b08e1cdd2836bdf2be612ac74b26d3b