Development of an amphotropic, high-titer retrovirus vector expressing the dihydrofolate reductase gene and conferring methotrexate resistance.
Altered mouse dihydrofolate reductase gene (DHFRR) was expressed in murine cells using Abelson murine leukemia provirus genome as a prototype vector. A cDNA clone of DHFRR was inserted into a plasmid structure containing retroviral transcriptional as well as packaging signals. The recombinant plasmid was transfected into psi-2 ecotropic cells and the transient virus was used to infect amphotropic PA-12 cells. Recombinant virus (ABL-DHFRR) was detected in the culture medium of transfected PA-12 cells and was free of helper virus. The ABL-DHFRR was capable of conferring methotrexate (MTX) resistance to a variety of cells in culture. The titer of ABL-DHFRR virus was at least tenfold higher than other DHFR retroviruses. The ABL-DHFRR virus titer was increased by selection at increasing concentrations of MTX. The presence of the DHFRR in the virus-infected cells was confirmed by assays which showed reduced inhibition of enzyme activity by MTX. A helper-virus-free, amphotropic, high-titer retrovirus containing the altered DHFR was obtained which may be of use as a dominant selectable marker in infecting hematopoietic progenitor cells.
D000011:Abelson murine leukemia virus / Q000235:genetics*; D000818:Animals; D003001:Cloning, Molecular; D004351:Drug Resistance; D005822:Genetic Vectors; D006378:Helper Viruses / Q000235:genetics; D009052:Leukemia Virus, Murine / Q000235:genetics*; D008727:Methotrexate / Q000494:pharmacology; D051379:Mice; D010957:Plasmids; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*
D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate