RESUMO
The coordinate expression of a marker gene and therapeutic genes in a single vector is markedly advantageous. The internal ribosome entry site (IRES) from encephalomyocarditis and that from polio viruses were used to construct a polycistronic retroviral vector pGCEN/TRI, where Neo-resistant gene was used as a marker gene and TNF-alpha cDNA, IL-2 cDNA as genes of interest, so that the LTR promoter derived the expression of a tricistronic mRNA. Amphotropic packaging cells PA317 transfected with pGCEN/TRI using LipofectAMINE was selected with G418 and the positive clones expressing the genes of interest to produce high-titer retrovirus (5x10(5) CFU/ml) were obtained. PCR confirmed the presence of proviral DNA in the positive producer cells and Northern blot analysis revealed a single, LTR promoted transcript. The transduced cells expressed TNF-alpha and IL-2 at different levels. This demonstrated that polycistronic retroviral vector containing IRES could efficiently express multiple therapeutic genes in the same target cell.
