Co-expression and amplification of dihydrofolate reductase cDNA and the Escherichia coli XGPRT gene in Chinese hamster ovary cells.
We have transformed Chinese hamster ovary cells with a plasmid containing mouse dihydrofolate reductase (DHFR) cDNA and the Escherichia coli xanthine-guanine phosphoribosyl transferase (XGPRT) gene under the control of the mouse mammary tumor virus and SV40 early promoters, respectively. Selection for the expression of XGPRT using the dominant selection scheme described by Mulligan and Berg yields clones that simultaneously express DHFR. Growth of these cells in progressively increasing concentrations of methotrexate, results in selection of cells that overproduce DHFR and its messenger RNA 250-500 fold. ANalyses of the plasmid DNA sequences in these cells reveal that the increased production of DHFR is due in part to gene amplification (approximately 50-fold) and in part to a selective overproduction of DHFR RNA. Last, the methotrexate-resistant cells contain 50 times more XGPRT RNA and DNA than the initial transformant; this demonstrates the potential for using gene amplification as a means for overproducing the products of genes linked to DHFR cDNA in plasmid vectors.
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000235:genetics*; D004351:Drug Resistance; D004926:Escherichia coli / Q000235:genetics*; D005260:Female; D005784:Gene Amplification; D005786:Gene Expression Regulation; D008727:Methotrexate / Q000494:pharmacology; D051379:Mice; D009693:Nucleic Acid Hybridization; D010053:Ovary; D010430:Pentosyltransferases / Q000235:genetics*; D010957:Plasmids; D012313:RNA / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000172:deficiency / Q000235:genetics*; D014170:Transformation, Genetic
D012313:RNA; D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase; D010430:Pentosyltransferases; C024827:xanthine phosphoribosyltransferase; D008727:Methotrexate