NDLI: Transfer of the bacterial gene for cytosine deaminase to mammalian cells confers lethal sensitivity to 5-fluorocytosine: a negative selection system.

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Transfer of the bacterial gene for cytosine deaminase to mammalian cells confers lethal sensitivity to 5-fluorocytosine: a negative selection system.

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Transfer of the bacterial gene for cytosine deaminase to mammalian cells confers lethal sensitivity to 5-fluorocytosine: a negative selection system.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Blaese, R. M. Mullen, C. A. Kilstrup, M.
Description	Author Affiliation: Mullen CA ( Cellular Immunology Section, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.);
Abstract	Expression of the bacterial gene for cytosine deaminase (CD; EC 3.5.4.1) in mammalian cells was evaluated as a negative selection system or suicide vector for potential use in gene transfer studies and therapies. Mammalian cells, unlike certain bacteria and fungi, do not contain the enzyme CD and do not ordinarily metabolize cytosine to uracil. Nor do they metabolize the innocuous compound 5-fluorocytosine to the highly toxic compound 5-fluorouracil. The Escherichia coli CD gene underwent PCR oligonucleotide-directed mutagenesis to enhance its expression in a eukaryotic system and it was then cloned into an expression vector, pLXSN, that also contains a neomycin-resistance gene. Murine fibroblast lines were transfected with the plasmid and subjected to brief selection in the neomycin analogue G418. Lysates from these cell populations exhibited significant CD activity detected by conversion of radiolabeled cytosine to uracil. In clonogenic assays transfected cells expressing CD were selectively killed by incubation in 5-fluorocytosine, whereas control cell lines were not. Dose-response studies evaluating [3H]thymidine incorporation or cloning efficiency demonstrated profound inhibition at and above 65 micrograms of 5-fluorocytosine per ml. Mixed cellular assays showed that CD-positive cells could be eliminated without bystander killing of other cells. Retrovirus-mediated CD gene transfer into various tissues was also demonstrated. Thus CD, with its ability to produce the toxic antimetabolite 5-fluorouracil from 5-fluorocytosine, may be useful as a negative selection system for studies and treatments employing gene transfer techniques.
ISSN	00278424
e-ISSN	10916490
Journal	Proceedings of the National Academy of Sciences of the United States of America
Issue Number	1
Volume Number	89
Language	English
Publisher	National Academy of Sciences
Publisher Date	1992-02-01
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Cells, Cultured Drug Effects Flucytosine Toxicity Genes, Bacterial Nucleoside Deaminases Genetics Animals Cloning, Molecular Cytosine Deaminase DNA Mutational Analysis Escherichia Coli Genetic Therapy Genetic Vectors In Vitro Techniques Mice Molecular Sequence Data Oligodeoxyribonucleotides Chemistry Transfection Multidisciplinary
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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