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Transfection of DHFR − and DHFR + mammalian cells using methotrexate‐resistant mutants of mouse dihydrofolate reductase
Author(s) -
Thillet Joëlle,
Pictet Raymond
Publication year - 1990
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(90)81213-8
Subject(s) - dihydrofolate reductase , mutant , transfection , microbiology and biotechnology , mutagenesis , methotrexate , biology , enzyme , chinese hamster ovary cell , wild type , antifolate , cell culture , gene , biochemistry , antimetabolite , genetics , immunology
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 transfert 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 μM methotrexate is correlated with the K i (MTX) of the enzyme: the higher the K i , the higher the number of colonies for the corresponding mutant. In contrast, the transfections of DHFR + cells gave a few numbers of colonies not different for the wild type and the mutants.