Ecotropic Cell Research Articles

Molecular Biology Techniques Q&A

Molecular Biology Techniques Q&A

Development of an amphotropic, high-titer retrovirus vector expressing the dihydrofolate reductase gene and conferring methotrexate resistance

Altered mouse dihydrofolate reductase gene ( DHFR R) was expressed in murine cells using Abelson murine leukemia provirus genome as a prototype vector. A cDNA clone of DHFR R was inserted into a plasmid structure containing retroviral transcriptional as well as packaging signals. The recombinant plasmid was transfected into ψ-2 ecotropic cells and the transient virus was used to infect amphotropic PA-12 cells. Recombinant virus ( ABL-DHFR R) was detected in the culture medium of transfected PA-12 cells and was free of helper virus. The ABL-DHFR R was capable of conferring methotrexate (MTX) resistance to a variety of cells in culture. The titer of ABL-DHFR R virus was at least tenfold higher than other DHFR retroviruses. The ABL-DHFR R virus titer was increased by selection at increasing concentrations of MTX. The presence of the DHFR R 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.