Transfection with a cDNA encoding a Ser31 or Ser34 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 vector carrying human dihydrofolate reductase-encoding cDNAs (DHFR) containing a Ser31 or a Ser34 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 polymerase chain reaction amplifications confirmed that the transfected plasmids were integrated into the CHO DNA. Gene copy number analysis revealed that both the Ser3 1 and the Ser3.4 mutants amplifiable when grown in increasing concentrations of MTX. Retrovirus-mediated gene transfer of the Ser31 mutant into mouse marrow progenitor cells also resulted in MTX-resistant CFU-GM (colony-forming unit-granulocyte macrophage) cells.
Banerjee
D
D
Molecular Pharmacology and Experimental Therapeutics Program, Memorial Sloan Kettering Cancer Center, New York, NY 10021.
Schweitzer
B I
BI
Volkenandt
M
M
Li
M X
MX
Waltham
M
M
Mineishi
S
S
Zhao
S C
SC
Bertino
J R
JR
D000818:Animals; D001120:Arginine / Q000235:genetics; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D001853:Bone Marrow / Q000378:metabolism*; D016466:CHO Cells; D003114:Colony-Forming Units Assay; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics*; D004351:Drug Resistance / Q000235:genetics; D005784:Gene Amplification; D018014:Gene Transfer Techniques; D005819:Genetic Markers; D006801:Humans; D008727:Methotrexate / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D012190:Retroviridae / Q000235:genetics; D012694:Serine / Q000235:genetics*; D013234:Stem Cells / Q000378:metabolism*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D014162:Transfection / Q000235:genetics
D018076:DNA, Complementary; D005819:Genetic Markers; D012694:Serine; D001120:Arginine; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate