Construction and use of a dominant, selectable marker: a Harvey sarcoma virus-dihydrofolate reductase chimera.
The transcriptional promoter of the Harvey sarcoma virus long terminal repeat has been used to construct a biologically active dihydrofolate reductase chimera. The construction placed the long terminal repeat at the 5' end of a dihydrofolate reductase cDNA. This chimera mediated methotrexate resistance when introduced into wild-type NIH3T3 mouse cells by transfection. The chimeric sequences were expressed in the form of polyadenylated RNA and dihydrofolate reductase protein and were amplified when the methotrexate-resistant transfectants were selected to grow in increasing methotrexate concentrations. This chimera was dominant acting and able to confer a methotrexate-resistant phenotype on wild-type NIH3T3 cells. It has been used in cotransfection experiments with DNA from human tumor cells to obtain foci of methotrexate-resistant transformed NIH3T3 cells resulting from uptake of exogenous DNA. The transfected methotrexate-resistant cells carried double minute chromosomes that appeared to contain DNA acquired during transfection.
D000818:Animals; D002478:Cells, Cultured; D002678:Chimera; D004274:DNA, Recombinant; D004352:Drug Resistance, Microbial; D005784:Gene Amplification; D005819:Genetic Markers; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000235:genetics; D008727:Methotrexate / Q000494:pharmacology; D051379:Mice; D009876:Operon; D012091:Repetitive Sequences, Nucleic Acid; D009053:Sarcoma Viruses, Murine / Q000235:genetics*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D014162:Transfection
D004274:DNA, Recombinant; D005819:Genetic Markers; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate