Expression of the plasmid-encoded type I dihydrofolate reductase gene in cultured mammalian cells: a novel selectable marker.
A recombinant plasmid has been designed to express the gene encoding a type I methotrexate-resistant dihydrofolate reductase, derived from the bacterial plasmid R483, in DHFR- Chinese hamster ovary cells. Vectors containing the wild type gene, whose coding sequence initiates with a GTG codon, fail to direct the synthesis of detectable levels of protein. Substitution of the GTG codon by an AG codon using in vitro mutagenesis overcomes this block; cells transfected with the modified vector synthesize a functional prokaryotic protein that sustains the growth of these cells in the presence of dihydrofolic acid in the culture media. This property is consistent with the inability of the type I enzyme to reduce folate to dihydrofolate, and enabled the development of a selection strategy whereby prokaryotic and mammalian DHFRs genes could be used sequentially as independently selectable markers.
Simonsen
C S
CS
Department of Molecular Biology, Genentech, Inc., San Francisco, CA 94080.
Walter
M
M
Levinson
A D
AD
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D006224:Cricetinae; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D005822:Genetic Vectors; D008727:Methotrexate / Q000494:pharmacology; D010957:Plasmids; D012641:Selection, Genetic; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D014162:Transfection
D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate