CONSTRUCTION AND CHARACTERIZATION OF SINGLE-TRANSCRIPT TRICISTRONIC RETROVIRAL VECTORS USING 2 INTERNAL RIBOSOME ENTRY SITES

We describe a series of retroviral vectors containing two internal rib osome entry sites (IRES) for the co-transcription of three genes. Tran scription of the single-transcript tricistronic mRNA is under the cont rol of a Harvey murine sarcoma virus long terminal repeat. The 5'-most open reading frame is under either cap-dependent or cap-independent t ranslational control, while the two downstream open reading frames are translated in a cap-independent fashion using the initiation codons o f their respective IRES elements. Both IRES elements are taken from th e encephalomyocarditis virus. To characterize these vectors, we used t he human multidrug resistance gene (MDR1) in the 5' position, the gene for green fluorescent protein (GFP) in the middle position, and neo i n the 3' position. The vectors were either transfected directly into N IH3T3 mouse fibroblasts or packaged into retrovirus and then transduce d into NIH3T3 cells. Gene transfer was followed by selection with colc hicine, which selects for expression of the MDR1 gene, or with G418, w hich selects for expression of the neo gene. Thus, we could determine the function of the tricistronic vectors under conditions of selection for either the 5'-most or the 3'-most gene. In DNA-mediated transfect ions, we were able to achieve expression of all three open reading fra mes lander either selection condition, We obtained higher expression o f all three genes when colchicine was used to select for MDR1 expressi on than when G418 was used to select for neo expression. Expression of the non-selected GFP gene (the middle cistron) was unstable, most lik ely due to loss of integrated GFP DNA sequences during long-term cultu ring. We were able to achieve retrovirus-mediated transduction of all three genes, bur this was an inefficient process.