Transfection of DHFR- and DHFR+ mammalian cells using methotrexate-resistant mutants of mouse dihydrofolate reductase.
Site-directed mutagenesis was used to generate mutants of mouse dihydrofolate reductase more resistant to methotrexate than the wild type enzyme. The mutant genes were used to transfect either DHFR- or DHFR+ cell lines. These mutants, as well as the wild type gene, were able to confer methotrexate resistance to DHFR- CHO cells. The number of selected colonies decreased with increased concentrations of methotrexate. The number of colonies observed at 10 microM methotrexate is correlated with the Ki(MTX) of the enzyme: the higher the Ki, the higher the number of colonies for the corresponding mutant. In contrast, the transfection of DHFR+ cells gave a few numbers of colonies not different for the wild type and the mutants.
Institut Jacques Monod, Unité INSERM 257, Université Paris VI.
J
J
Thillet
R
R
Pictet
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004351:Drug Resistance / Q000235:genetics; D005260:Female; D007700:Kinetics; D007739:L Cells / Q000201:enzymology; D008727:Methotrexate / Q000494:pharmacology*; D051379:Mice; D009154:Mutation; D010053:Ovary; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate