兰州大学机构库
Developing a Versatile Shotgun Cloning Strategy for Single-Vector-Based Multiplex Expression of Short Interfering RNAs (siRNAs) in Mammalian Cells
2019-09
Source PublicationACS Synthetic Biology   Impact Factor & Quartile Of Published Year  The Latest Impact Factor & Quartile
ISSN2161-5063
Volume8Issue:9Pages:2092-2105
page numbers27
AbstractAs 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.
KeywordRNA 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
PublisherAMER CHEMICAL SOC
DOI10.1021/acssynbio.9b00203
Indexed BySCIE
Language英语
Funding ProjectNational Key Research and Development Program of China[2016YFC1000803][2011CB707906] ; National Institutes of Health[CA226303] ; U.S. Department of Defense[OR130096] ; University of Chicago Cancer Center Support Grant[P30CA014599] ; National Center for Advancing Translational Sciences of the National Institutes of Health[UL1 TR000430]
WOS Research AreaBiochemistry & Molecular Biology
WOS SubjectBiochemical Research Methods
WOS IDWOS:000487577300015
PublisherAMER CHEMICAL SOC
Original Document TypeArticle
PMID 31465214
Citation statistics
Document Type期刊论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/416696
Collection兰州大学
第二临床医学院
Corresponding AuthorHe, Tong-Chuan; Zeng, Zongyue
Affiliation
1.Chongqing Med Univ, Key Lab Diagnost Med, Minist Educ, Chongqing, Peoples R China
2.Chongqing Med Univ, Sch Lab & Diagnost Med, Chongqing, Peoples R China
3.Univ Chicago, Med Ctr, Dept Orthopaed Surg & Rehabil Med, Mol Oncol Lab, Chicago, IL 60637 USA
4.China Three Gorges Univ, Sch Med, Dept Biochem & Mol Biol, Yichang, Peoples R China
5.Nanchang Univ, Affiliated Hosp 2, Dept Clin Lab Med, Nanchang 330006, Jiangxi, Peoples R China
6.Chongqing Med Univ, Affiliated Hosp, Chongqing 400016, Peoples R China
7.Lanzhou Univ, Key Lab Orthopaed Surg Gansu Prov, Lanzhou 730030, Gansu, Peoples R China
8.Soochow Univ, Affiliated Hosp 2, Dept Gen Surg, Suzhou 215004, Peoples R China
9.Chongqing Gen Hosp, Dept Orthopaed Surg, Chongqing 400013, Peoples R China
10.Qingdao Univ, Affiliated Hosp, Dept Clin Lab Med, Qingdao, Shandong, Peoples R China
11.Southwest Univ, Sch Life Sci, Chongqing 400715, Peoples R China
12.Sichuan Univ, West China Hosp, Dept Burn & Plast Surg, Chengdu 610041, Sichuan, Peoples R China
13.Guangzhou Univ Chinese Med, Affiliated Hosp 1, Dept Orthopaed Surg, Guangzhou 510405, Guangdong, Peoples R China
Recommended Citation
GB/T 7714
Wang, Xi,Yuan, Chengfu,Huang, Bo,et al. Developing a Versatile Shotgun Cloning Strategy for Single-Vector-Based Multiplex Expression of Short Interfering RNAs (siRNAs) in Mammalian Cells[J]. ACS Synthetic Biology,2019,8(9):2092-2105.
APA Wang, Xi.,Yuan, Chengfu.,Huang, Bo.,Fan, Jiaming.,Feng, Yixiao.,...&Zeng, Zongyue.(2019).Developing a Versatile Shotgun Cloning Strategy for Single-Vector-Based Multiplex Expression of Short Interfering RNAs (siRNAs) in Mammalian Cells.ACS Synthetic Biology,8(9),2092-2105.
MLA Wang, Xi,et al."Developing a Versatile Shotgun Cloning Strategy for Single-Vector-Based Multiplex Expression of Short Interfering RNAs (siRNAs) in Mammalian Cells".ACS Synthetic Biology 8.9(2019):2092-2105.
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