High-throughput gateway bicistronic retroviral vectors for stable expression in mammalian cells: exploring the biologic effects of STAT5 overexpression.
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 expression of GFP, which was cloned downstream of the IRES. We used the same approach and generated Ba/F3 cells that overexpress STAT5a, STAT5b, or a constitutively active form of STAT5. Overexpression of STAT5 lead to a significant effect on the intrinsic adherence to plastic of these cells, but did not change their proliferative responses to cytokines. We discuss possible applications of the new vectors for cell signaling and expression cloning.
Royer
Yohan
Y
Ludwig Institute for Cancer Research, Brussels, Belgium.
Menu
Catherine
C
Liu
Xuedong
X
Constantinescu
Stefan N
SN
D000818:Animals; D015153:Blotting, Western; D002448:Cell Adhesion / Q000502:physiology; D002478:Cells, Cultured; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D049452:Green Fluorescent Proteins; D053614:Janus Kinase 2; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008894:Milk Proteins; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D017467:Receptors, Erythropoietin / Q000235:genetics / Q000378:metabolism*; D012190:Retroviridae / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000235:genetics; D050799:STAT5 Transcription Factor; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014161:Transduction, Genetic / Q000379:methods*
D017931:DNA Primers; D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D008894:Milk Proteins; D011518:Proto-Oncogene Proteins; D017467:Receptors, Erythropoietin; D050799:STAT5 Transcription Factor; C494093:Stat5a protein, mouse; C494096:Stat5b protein, mouse; D015534:Trans-Activators; D049452:Green Fluorescent Proteins; D011505:Protein-Tyrosine Kinases; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2