TRANSFECTION WITH A CDNA-ENCODING A SER(31) OR SER(34) MUTANT HUMAN DIHYDROFOLATE-REDUCTASE INTO CHINESE-HAMSTER OVARY AND MOUSE MARROW PROGENITOR CELLS CONFERS METHOTREXATE RESISTANCE

Chinese hamster ovary (CHO) DHFR(-) cells were converted into the DHFR (+) phenotype when they were transfected with a mammalian expression v ector carrying human dihydrofolate reductase-encoding cDNAs (DHFR) con taining a Ser(31) or a Ser(34) mutation. Furthermore, transfection of these mutants into wild-type CHO cells resulted in resistance to high levels of methotrexate (MTX), indicating that these human variants can act as dominant selectable markers. Southern blot analysis and polyme rase chain reaction amplifications confirmed that the transfected plas mids were integrated into the CHO DNA. Gene copy number analysis revea led that both the Ser(31) and the Ser(34) mutants are amplifiable when grown in increasing concentrations of MTX. Retrovirus-mediated gene t ransfer of the Ser(31) mutant into mouse marrow progenitor cells also resulted in MTX-resistant CFU-GM (colony-forming unit-granulocyte macr ophage) cells.