Use of the Escherichia coli chromosomal DHFR gene as selection marker in mammalian cells.
The folA gene, the chromosomal dhfr gene of Escherichia coli, was engineered for expression in mammalian cells. In contrast to plasmid-derived bacterial dhfr genes previously used as selection markers in mammalian cells, the folA gene product is inhibitable by methotrexate (MTX) and trimethoprim (TMP). Therefore, this dhfr may present an alternative to mammalian dhfr species currently used as amplifiable selection markers. Transfected E. coli folA dhfr could complement the lack of endogenous DHFR in Chinese hamster ovary (CHO) cells lacking a functional dhfr gene. Both MTX and TMP inhibited growth of E. coli folA dhfr-transfected CHO cells. Expression of E. coli folA DHFR could be visualized by incubating the transfected cells with a fluorescent methotrexate derivative (F-MTX). Binding of F-MTX to E. coli folA DHFR was inhibitable as by both MTX and TMP, whereas MTX but not TMP blocked binding of F-MTX to recombinant mouse DHFR.
Asselbergs
F A
FA
Pharma Research Department, CIBA-GEIGY Ltd., Basel, Switzerland.
Widmer
R
R
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003587:Cytomegalovirus / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005434:Flow Cytometry; D015870:Gene Expression / Q000187:drug effects; D005798:Genes, Bacterial; D005819:Genetic Markers; D005822:Genetic Vectors; D008727:Methotrexate / Q000494:pharmacology; D051379:Mice; D011401:Promoter Regions, Genetic; D011817:Rabbits; D015183:Restriction Mapping; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D014162:Transfection; D014295:Trimethoprim / Q000494:pharmacology
D005819:Genetic Markers; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate