Available Vector Systems
Retroviral Vectors:
The retroviral vector of choice of the VCF is a vector constructed by Drs. Paul Robbins and Braydon Guild while they were postdoctoral fellows in the laboratory of Dr. Richard Mulligan. This vector, termed MFG, is a simplified vector that does not carry a selectable marker gene. This vector has been used extensively by Dr. Mulligan's laboratory and by UPCI investigators, and it has been shown to give high levels of expression of a variety of inserted genes following non-selective infection. The MFG vector contains a portion of the viral gag protein (half gag) up to the Narl site at post 1046. A fragment containing the splice acceptor (XhoI to Xbal) from MLV was inserted downstream of the Narl site. A synthetic oligonucleotide was used to introduce a Ncol site at the normal envelope ATG. A BarnH1 site was inserted just downstream from the Ncol site so that the cDNA of interest could be inserted using the unique Ncol and BamH1 cloning sites. Thus, therapeutic cDNAs inserted into MFG are expressed off a spliced message that resembles the authentic env message from MLV. Translation of the spliced message appears to be very efficient since it uses the normal env translation signals. To decrease the possibility of helper virus arising by recombination, a SacII linker was inserted just downstream of the gag ATG, introducing a frameshift so that there is no translation of the gag protein.
Expression of various cytokines, receptors, enzymes, and growth factors has been extremely high when inserted into MFG. Because of the high titer and robust expression mediated by MFG of inserted genes, it was of interest to determine if similar, high-level expression can be obtain of two genes in MFG. The ability to express two genes efficiently in a MFG-based vector will allow for the generation of safe "suicide" vectors using the HSV tk gene. To introduce multiple genes into MFG, the IRES sequence from encephalomyocarditis (EMCV) that allows translation of a downstream gene in a polycistronic message was utilized. This sequence has been shown to function efficiently in the context of a retroviral vector to express two genes from a polycistronic message. The MFG-IRES vectors were shown to efficiently express two genes from the same message using two different secreted marker proteins, IL1Ra (IRAP) and growth hormone. The MFG-IRES vectors have now been used to express three genes such as both subunits of IL-12 as well as a neor marker gene using two IRES elements.