Construction of a dominant selectable marker using a novel dihydrofolate reductase.
Simian virus 40 promoter-enhancer-based mammalian expression plasmids using dihydrofolate reductase (DHFR)-encoding cDNA sequences originally isolated from two methotrexate (MTX)-resistant, DHFR-overproducing Chinese hamster lung cell lines were constructed. One, designated pSVA75, contains a DHFR cDNA that encodes leucine (Leu22) and corresponds to the wild type (wt), MTX-sensitive form of the enzyme [Melera et al., J. Biol. Chem. 263 (1988) 1978-1990]. The other plasmid, pSVA3, contains a cDNA that encodes a novel mutant form of the enzyme in which Leu22 has been changed to Phe [Melera et al., Mol. Cell Biol. 4 (1984) 38-48]. The resulting DHFR displays a 20-fold-enhanced resistance to inhibition by MTX, but maintains the catalytic activity of the wt enzyme [Albrecht et al., Cancer Res. 32 (1972) 1539-1546]. Transfection of DHFR- Chinese hamster ovary cells with either plasmid demonstrated that both were able to reconstitute the DHFR+ phenotype with equal efficiency (i.e., greater than 2.5 x 10(-3), indicating that both the wt and mutant enzymes were catalytically active in transfected cells. In addition, the mutant form of the enzyme was found to act as a dominant selectable marker when transfected into diploid DHFR+ cells, and to allow selection of resistant clones at low MTX concentrations (125 nM MTX) with a frequency of greater than 8 x 10(-4). Moreover, transfected clones were found to amplify their exogenous DHFR sequences to reasonably high levels (42-fold) at relatively low (888 nM) MTX concentrations, suggesting that substantial amplification of DHFR DNA and cotransfected sequences as well, can be achieved with this vector.
Hussain
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A
Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201.
Lewis
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D
Yu
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Melera
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PW
D000818:Animals; D015139:Blotting, Southern; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D005799:Genes, Dominant / Q000235:genetics*; D005819:Genetic Markers / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D014162:Transfection / Q000235:genetics
D005819:Genetic Markers; D013762:Tetrahydrofolate Dehydrogenase