Transfection of a nonactive site mutant murine DHFR cDNA (the tryptophan 15 mutant) into Chinese hamster ovary and mouse marrow progenitor cells imparts MTX resistance in vitro.
Chinese hamster ovary (CHO) DHFR-cells were converted to the DHFR+ (dihydrofolate reductase) phenotype when transfected with a mammalian expression vector containing the murine mutant Trp15 DHFR cDNA. Transfection of the Trp15 DHFR cDNA into wild-type CHO cells resulted in resistance to high levels of methotrexate (MTX) in vitro indicating that this mutant DHFR cDNA can act as a dominant marker. Southern and Northern blot analyses of transfected cells indicated that the transfected mutant DHFR cDNA was integrated and expressed. Gene copy number analysis showed that the Trp15 cDNA was amplifiable in increasing concentrations of MTX. Transfection of murine bone marrow progenitor cells with the Trp15 mutant DHFR cDNA resulted in MTX resistant colony forming units-granulocyte macrophage.
Banerjee
D
D
Molecular Pharmacology and Therapeutics Program, Sloan Kettering Institute for Cancer Research, New York, NY 10021, USA.
Zhao
S C
SC
Tong
Y
Y
Steinherz
J
J
Gritsman
K
K
Bertino
J R
JR
D000818:Animals; D001854:Bone Marrow Cells; D016466:CHO Cells / Q000187:drug effects* / Q000378:metabolism; D003114:Colony-Forming Units Assay; D006224:Cricetinae; D003412:Cricetulus; D018076:DNA, Complementary / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics; D005784:Gene Amplification; D006412:Hematopoietic Stem Cells / Q000187:drug effects* / Q000378:metabolism; D008727:Methotrexate / Q000494:pharmacology*; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate