Transfection of DHFR- and DHFR+ mammalian cells using methotrexate-resistant mutants of mouse dihydrofolate reductase |
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Authors: | J Thillet R Pictet |
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Institution: | Institut Jacques Monod, Unité INSERM 257, Université Paris VI. |
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Abstract: | 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. |
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