Document Detail


Generation of a stable mammalian cell line for simultaneous expression of multiple genes by using 2A peptide-based lentiviral vector.
MedLine Citation:
PMID:  19034387     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Generation of mammalian cells stably expressing multiple exogenous genes is currently difficult. Here we provide a strategy to facilitate this process. First, a helper vector p2A containing three coding sequences for viral 2A peptides was constructed. Three reporter genes coding for red fluorescent protein (DsRed), firefly luciferase (Fluc) and enhanced green fluorescent protein (EGFP) were then inserted into p2A to form a fusion open reading frame that was subsequently subcloned into a lentiviral vector. After transduction, EGFP-positive 293T cells were selected by fluorescence activated cell sorting. The expression of exogenous genes in selected cells was stable for more than 15 passages, and EGFP-positive cells were over 95%. The efficient cleavages of 2A-peptide mediated polyprotein were also observed and all three reporter proteins were functional. Thus, a stable DsRed/Fluc/EGFP-coexpressing cell line was readily established within a short time. The strategy could be useful for basic research and protein production.
Authors:
Tao Hu; Qiong Fu; Ping Chen; Ke Zhang; Deyin Guo
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2008-11-26
Journal Detail:
Title:  Biotechnology letters     Volume:  31     ISSN:  1573-6776     ISO Abbreviation:  Biotechnol. Lett.     Publication Date:  2009 Mar 
Date Detail:
Created Date:  2009-02-02     Completed Date:  2009-02-27     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8008051     Medline TA:  Biotechnol Lett     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  353-9     Citation Subset:  IM    
Affiliation:
State Key Laboratory of Virology and The Modern Virology Research Centre, College of Life Sciences, Wuhan University, Wuhan, 430072, People's Republic of China.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Artificial Gene Fusion
Base Sequence
Cell Culture Techniques / methods*
Cell Line
Gene Expression*
Genes, Reporter
Genetic Vectors*
Green Fluorescent Proteins / genetics,  metabolism
Humans
Lentivirus / genetics*,  growth & development
Luciferases, Firefly / genetics,  metabolism
Luminescent Proteins / genetics,  metabolism
Molecular Sequence Data
Recombinant Fusion Proteins / genetics,  metabolism
Recombinant Proteins / biosynthesis,  genetics
Chemical
Reg. No./Substance:
0/Luminescent Proteins; 0/Recombinant Fusion Proteins; 0/Recombinant Proteins; 0/enhanced green fluorescent protein; 0/red fluorescent protein; 147336-22-9/Green Fluorescent Proteins; EC 1.13.12.7/Luciferases, Firefly

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