Two dominant-acting selectable markers for gene transfer studies in mammalian cells - PubMed (original) (raw)

Two dominant-acting selectable markers for gene transfer studies in mammalian cells

S C Hartman et al. Proc Natl Acad Sci U S A. 1988 Nov.

Abstract

We report the development of two dominant-acting genetic markers useful for monitoring gene transfer in mammalian cells that are based on prokaryotic genes encoding key steps in the synthesis of the essential amino acids, tryptophan and histidine. Under appropriate conditions, expression of these genes obviates the nutritional requirements for their respective amino acid products. Expression of the trpB gene of Escherichia coli, which encodes the beta subunit of tryptophan synthase (EC 4.2.1.20), allows mammalian cell survival and multiplication in medium containing indole in place of tryptophan. The hisD gene of Salmonella typhimurium encodes histidinol dehydrogenase (EC 1.1.1.23), which catalyzes the two-step NAD+-dependent oxidation of L-histidinol to L-histidine. In medium lacking histidine and containing histidinol, only mammalian cells expressing the hisD product survive. The selection is a double one in that the provided precursor histidinol is itself toxic to animal cells through its inhibition of histidyl-tRNA synthetase; thus, the dehydrogenase both removes an inhibitor and forms a required end product. Alternatively, the his selection may be carried out under conditions in which the dehydrogenase serves mainly to detoxify histidinol. For either the trp or his selections the substitute nutrient (indole or histidinol) is readily available, inexpensive, stable, permeable to cells, and convertible to the end product in a step controlled by a single gene. Vectors based upon murine retrovirus and papovavirus backbones have been successfully employed for both genes, allowing selection in a range of cell types, including 3T3, CV-1, and HeLa. These dominant selective schemes should provide generally useful and inexpensive alternatives to others currently in use, such as the gpt, neo, hygro, dhfr, and tk selections.

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Two dominant-acting selectable markers for gene transfer studies in mammalian cells - PubMed (original) (raw)