title: pIRES-CD4t, a dicistronic expression vector for MACS- or FACS-based selection of transfected cells.
abstract: To facilitate the selection of cell lines expressing transfected genes of interest, a plasmid vector has been constructed that directs the co-expression of heterologous cDNAs and a 3'-positioned cassette encoding a truncated CD4 marker. An encephalomyocarditis virus internal ribosomal entry site (IRES) mediates translational initiation from this 3' cassette, and a cytomegalovirus promoter drives dicistronic transcript expression. To test the utility of this vector, a luciferase reporter gene was inserted, and this construct (pIRES-CD4t-luc) was electrotransfected into myeloid FDCW2 cells. As monitored by flow cytometry and luciferase assays, three rounds of magnetic cells sorting (MACS) yielded > or = 90% CD4t-positive cells with an average density of 17,000 CD4t molecules per cell. In ten clonal sublines analyzed, luciferase expression was uniformly high and stable over a test period of three months. Finally, a comparison of MACS- vs. FACS-based isolation of transfected cells showed two to three rapid rounds of MACS to be somewhat more effective. Thus, pIRES-CD4t should prove useful in the direct and rapid selection of relevant stably or transiently transfected cells.
authors.affiliation: Pennsylvania State University, University Park 16802, USA.
authors.forename: P
authors.initials: P
authors.lastname: Gaines
authors.forename: D M
authors.initials: DM
authors.lastname: Wojchowski
mesh_terms: D000818:Animals; D001709:Biotechnology; D015704:CD4 Antigens / Q000235:genetics; D002460:Cell Line; D002469:Cell Separation; D004686:Encephalomyocarditis virus / Q000235:genetics; D005434:Flow Cytometry; D015870:Gene Expression; D005796:Genes; D017930:Genes, Reporter; D005822:Genetic Vectors; D018189:Immunomagnetic Separation; D008156:Luciferases / Q000235:genetics; D051379:Mice; D014162:Transfection
chemical_list: D015704:CD4 Antigens; D008156:Luciferases