The use of a wild-type dihydrofolate reductase-encoding cDNA as a dominant selectable marker and induction of expression by methotrexate.
The cDNA (DHFR) encoding the wild-type (wt) dihydrofolate reductase (DHFR) was used as a dominant selectable marker in the transfection of murine hybridoma Sp2/0-Ag14 cells by protoplast fusion. The initial clones contained 100-400 copies of integrated plasmid DNA, and the high level of wt DHFR protein produced enabled the cells to survive the drug selection at 100 nM methotrexate (MTX). The expression of the gene of interest was several fold higher than when the mutant DHFR with decreased MTX binding was used as the selection marker, presumably because the clones were more sensitive to the stress induced by MTX. When the clones were propagated at higher concentrations of MTX, expression of both DHFR and the gene of interest increased. This induction is freely reversible, and we have shown that it is controlled at the transcriptional level, by nuclear run-off transcription assays.
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Abbott Biotech Inc., Needham Heights MA 02194.
Lynch
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Gillies
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D001483:Base Sequence; D004247:DNA / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors; D008727:Methotrexate / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D012333:RNA, Messenger / Q000235:genetics; D012641:Selection, Genetic; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection
D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate