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