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.
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References
de Felipe P, Martin V, Cortes ML, Ryan M, Izquierdo M (1999) Use of the 2A sequence from foot-and-mouth disease virus in the generation of retroviral vectors for gene therapy. Gene Ther 6:198–208
DeMaria CT et al (2007) Accelerated clone selection for recombinant CHO CELLS using a FACS-based high-throughput screen. Biotechnol Prog 23:465–472
Donnelly ML et al (2001) Analysis of the aphthovirus 2A/2B polyprotein “cleavage” mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal “skip”. J Gen Virol 82:1013–1025
Emerman M, Temin HM (1984) Genes with promoters in retrovirus vectors can be independently suppressed by an epigenetic mechanism. Cell 39:449–467
Gaken J et al (2000) Fusagene vectors: a novel strategy for the expression of multiple genes from a single cistron. Gene Ther 7:1979–1985
Hasegawa K, Cowan AB, Nakatsuji N, Suemori H (2007) Efficient multicistronic expression of a transgene in human embryonic stem cells. Stem Cells 25:1707–1712
Klump H, Schiedlmeier B, Vogt B, Ryan M, Ostertag W, Baum C (2001) Retroviral vector-mediated expression of HoxB4 in hematopoietic cells using a novel coexpression strategy. Gene Ther 8:811–817
Lengler J, Holzmuller H, Salmons B, Gunzburg WH, Renner M (2005) FMDV-2A sequence and protein arrangement contribute to functionality of CYP2B1-reporter fusion protein. Anal Biochem 343:116–124
Li Z et al (2005) Specific inhibition of HIV-1 replication by short hairpin RNAs targeting human cyclin T1 without inducing apoptosis. FEBS Lett 579:3100–3106
Meng YG, Liang J, Wong WL, Chisholm V (2000) Green fluorescent protein as a second selectable marker for selection of high producing clones from transfected CHO cells. Gene 242:201–207
Mizuguchi H, Xu Z, Ishii-Watabe A, Uchida E, Hayakawa T (2000) IRES-dependent second gene expression is significantly lower than cap-dependent first gene expression in a bicistronic vector. Mol Ther 1:376–382
Pfeifer A, Ikawa M, Dayn Y, Verma IM (2002) Transgenesis by lentiviral vectors: lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos. Proc Natl Acad Sci USA 99:2140–2145
Rubinson DA et al (2003) A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet 33:401–406
Ryan MD, Drew J (1994) Foot-and-mouth disease virus 2A oligopeptide mediated cleavage of an artificial polyprotein. EMBO J 13:928–933
Santhosh CV, Tamhane MC, Kamat RH, Patel VV, Mukhopadhyaya R (2008) A lentiviral vector with novel multiple cloning sites: stable transgene expression in vitro and in vivo. Biochem Biophys Res Commun 371:546–550
Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2:905–909
Stemmer WP, Crameri A, Ha KD, Brennan TM, Heyneker HL (1995) Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene 164:49–53
Suter DM et al (2006) Rapid generation of stable transgenic embryonic stem cell lines using modular lentivectors. Stem Cells 24:615–623
Szymczak AL et al (2004) Correction of multi-gene deficiency in vivo using a single “self-cleaving” 2A peptide-based retroviral vector. Nat Biotechnol 22:589–594
Yu X et al (2003) Lentiviral vectors with two independent internal promoters transfer high-level expression of multiple transgenes to human hematopoietic stem-progenitor cells. Mol Ther 7:827–838
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This study was supported by China “863” programme (grant 2006AA02Z123) and the MOE “111” project #B06018.
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Hu, T., Fu, Q., Chen, P. et al. Generation of a stable mammalian cell line for simultaneous expression of multiple genes by using 2A peptide-based lentiviral vector. Biotechnol Lett 31, 353–359 (2009). https://doi.org/10.1007/s10529-008-9882-3
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DOI: https://doi.org/10.1007/s10529-008-9882-3