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.
