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.[1]References
- Construction and use of a dominant, selectable marker: a Harvey sarcoma virus-dihydrofolate reductase chimera. Murray, M.J., Kaufman, R.J., Latt, S.A., Weinberg, R.A. Mol. Cell. Biol. (1983) [Pubmed]
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