title: Generation of a stable mammalian cell line for simultaneous expression of multiple genes by using 2A peptide-based lentiviral vector.
abstract: 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.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.
authors.forename: Tao
authors.initials: T
authors.lastname: Hu
authors.forename: Qiong
authors.initials: Q
authors.lastname: Fu
authors.forename: Ping
authors.initials: P
authors.lastname: Chen
authors.forename: Ke
authors.initials: K
authors.lastname: Zhang
authors.forename: Deyin
authors.initials: D
authors.lastname: Guo
mesh_terms: D000595:Amino Acid Sequence; D019677:Artificial Gene Fusion; D001483:Base Sequence; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics* / Q000254:growth & development; D049409:Luciferases, Firefly / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D000097573:Red Fluorescent Protein
chemical_list: D049452:Green Fluorescent Proteins; D049409:Luciferases, Firefly; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000097573:Red Fluorescent Protein; C485184:enhanced green fluorescent protein