A simplified and versatile system for the simultaneous expression of multiple siRNAs in mammalian cells using Gibson DNA Assembly.
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.
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.
Fang
F
Deng
Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, 60637, United States of America.
Xiang
X
Chen
Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, 60637, United States of America; Department of Orthopaedic Surgery, the Affiliated Xiang-Ya Hospital of Central South University, Changsha, 410008, China.
Zhan
Z
Liao
Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, 60637, United States of America; Ministry of Education Key Laboratory of Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing, 400016, China.
Zhengjian
Z
Yan
Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, 60637, United States of America; Ministry of Education Key Laboratory of Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing, 400016, China.
Zhongliang
Z
Wang
Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, 60637, United States of America; Ministry of Education Key Laboratory of Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing, 400016, China.
Youlin
Y
Deng
Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, 60637, United States of America; Ministry of Education Key Laboratory of Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing, 400016, China.
Qian
Q
Zhang
Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, 60637, United States of America; Department of Surgery, the Affiliated Zhongnan Hospital of Wuhan University, Wuhan, 430071, China.
Zhonglin
Z
Zhang
Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, 60637, United States of America; School of Bioengineering, Chongqing University, Chongqing, 400044, China.
Jixing
J
Ye
Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, 60637, United States of America; Ministry of Education Key Laboratory of Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing, 400016, China; Department of Orthopaedic Surgery, the Affiliated Xiang-Ya Hospital of Central South University, Changsha, 410008, China.
Min
M
Qiao
Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, 60637, United States of America; Department of Surgery, the Affiliated Zhongnan Hospital of Wuhan University, Wuhan, 430071, China.
Ruifang
R
Li
Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, 60637, United States of America.
Sahitya
S
Denduluri
Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, 60637, United States of America; Ministry of Education Key Laboratory of Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing, 400016, China.
Jing
J
Wang
Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, 60637, United States of America; Ministry of Education Key Laboratory of Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing, 400016, China.
Qiang
Q
Wei
Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, 60637, United States of America.
Melissa
M
Li
Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, 60637, United States of America.
Nisha
N
Geng
Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, 60637, United States of America; Department of Orthopaedic Surgery, the Second Affiliated Hospital of Lanzhou University, Lanzhou, Gansu, 730000, China.
Lianggong
L
Zhao
Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, 60637, United States of America.
Guolin
G
Zhou
Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, 60637, United States of America; Ministry of Education Key Laboratory of Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing, 400016, China.
Penghui
P
Zhang
Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, 60637, United States of America.
Hue H
HH
Luu
Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, 60637, United States of America.
Rex C
RC
Haydon
Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, 60637, United States of America; The Laboratory of Craniofacial Biology, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, 60637, United States of America.
Russell R
RR
Reid
Department of Cell Biology, Third Military Medical University, Chongqing, 400038, China.
Tian
T
Yang
Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, 60637, United States of America; Department of Orthopaedic Surgery, the Affiliated Xiang-Ya Hospital of Central South University, Changsha, 410008, China.
Tong-Chuan
TC
He
D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism*; D055427:Growth Differentiation Factor 2; D055412:Growth Differentiation Factors / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism; D051379:Mice; D010012:Osteogenesis; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D060509:Wnt3A Protein / Q000378:metabolism; D051176:beta Catenin / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
C525887:GDF2 protein, human; D055427:Growth Differentiation Factor 2; D055412:Growth Differentiation Factors; D034741:RNA, Small Interfering; D060509:Wnt3A Protein; D051176:beta Catenin; D004247:DNA