High-throughput gateway bicistronic retroviral vectors for stable expression in mammalian cells: exploring the biologic effects of STAT5 overexpression

DNA and Cell Biology
Yohan RoyerStefan N Constantinescu

Abstract

Stable expression of cloned genes in mammalian cells has been achieved in the past by retroviral transduction using bicistronic retroviral vectors. In these vectors, the use of an Internal Ribosome Entry Site (IRES) allows simultaneous expression of a protein of interest and a fluorescence marker. However, traditional cDNA cloning in these vectors is often difficult. Here we report the construction of a high-throughput retroviral vector using the Invitrogen "Gateway" Cloning system. The Gateway recombination sequences (attR) flanking the ccdB and chloramphenicol resistance genes were incorporated at the 5' of the IRES of pMX-IRES-GFP, -CD2, or -CD4 vectors. Through recombination, these vectors can acquire cDNAs coding for genes of interest, which will result in simultaneous expression of the recombined gene and the marker protein. We constructed Gateway bicistronic vectors coding for the erythropoietin receptor (EpoR) and GFP, CD4, or CD2. Epo-dependent proliferation assays and analysis of Jak2-dependent EpoR cell-surface expression showed that these vectors were able to function indistinguishable from the original pMX-EpoR-IRES-GFP. The expression levels of the genes cloned upstream the IRES were proportional to the levels of ...Continue Reading

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Citations

Mar 14, 2007·The Journal of Experimental Medicine·Vincenzo Di BartoloOreste Acuto
Jul 13, 2006·Assay and Drug Development Technologies·Adriana BeltranPilar Blancafort
Mar 1, 2009·Expert Opinion on Drug Discovery·Robert S Ames, Quinn Lu
Sep 15, 2006·Analytical Biochemistry·Qi-Jun ChenXue-Chen Wang
Apr 7, 2015·Molecular Oncology·Monique G P van der WijstMarianne G Rots
Nov 26, 2008·Journal of Immunological Methods·Selinda J OrrDaniel W McVicar
Feb 13, 2014·New Biotechnology·Jeff Jia Cheng HouTrent P Munro
Mar 6, 2008·Molecular Cancer Therapeutics·Pilar BlancafortCarlos F Barbas

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