Selection against expression of the Escherichia coli gene gpt in hprt+ mouse teratocarcinoma and hybrid cells.

Thioxanthine is toxic for mammalian cells transformed by the dominant selectable marker gpt. It allowed us to select, in the presence of the endogenous hypoxanthine-guanine phosphoribosyltransferase gene, mutants that did not express gpt any more and also hybrid cells that had lost the chromosome carrying it. The gpt marker is thus dominant in negative as well as in positive selection, which makes it potentially very useful for genetic studies of mammalian cells.

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Optimization of the hygromycin B resistance-conferring gene as a dominant selectable marker in mammalian cells

Gene ◽

10.1016/0378-1119(90)90045-s ◽

1990 ◽

Vol 88 (2) ◽

pp. 285-288 ◽

Cited By ~ 19

Author(s):

Thomas J. Giordano ◽

William T. McAllister

Keyword(s):

Mammalian Cells ◽

Selectable Marker ◽

Hygromycin B ◽

Hygromycin B Resistance ◽

Dominant Selectable Marker

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Expression of Hygromycin Phosphotransferase Alters Virulence of Histoplasma capsulatum

Eukaryotic Cell ◽

10.1128/ec.00139-07 ◽

2007 ◽

Vol 6 (11) ◽

pp. 2066-2071 ◽

Cited By ~ 8

Author(s):

A. George Smulian ◽

Reta S. Gibbons ◽

Jeffery A. Demland ◽

Deborah T. Spaulding ◽

George S. Deepe

Keyword(s):

Mammalian Cells ◽

Histoplasma Capsulatum ◽

Fluorescent Protein ◽

Selectable Marker ◽

Animal Experiments ◽

Yeast Cells ◽

Growth And Survival ◽

Hygromycin Phosphotransferase ◽

Dominant Selectable Marker

ABSTRACT The Escherichia coli hygromycin phosphotransferase (hph) gene, which confers hygromycin resistance, is commonly used as a dominant selectable marker in genetically modified bacteria, fungi, plants, insects, and mammalian cells. Expression of the hph gene has rarely been reported to induce effects other than those expected. Hygromycin B is the most common dominant selectable marker used in the molecular manipulation of Histoplasma capsulatum in the generation of knockout strains of H. capsulatum or as a marker in mutant strains. hph-expressing organisms appear to have no defect in long-term in vitro growth and survival and have been successfully used to exploit host-parasite interaction in short-term cell culture systems and animal experiments. We introduced the hph gene as a selectable marker together with the gene encoding green fluorescent protein into wild-type strains of H. capsulatum. Infection of mice with hph-expressing H. capsulatum yeast cells at sublethal doses resulted in lethality. The lethality was not attributable to the site of integration of the hph construct into the genomes or to the method of integration and was not H. capsulatum strain related. Death of mice was not caused by altered cytokine profiles or an overwhelming fungal burden. The lethality was dependent on the kinase activity of hygromycin phosphotransferase. These results should raise awareness of the potential detrimental effects of the hph gene.

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Reversion analysis of mutations induced by 5-bromodeoxyuridine mutagenesis in mammalian cells.

Molecular and Cellular Biology ◽

10.1128/mcb.5.11.3092 ◽

1985 ◽

Vol 5 (11) ◽

pp. 3092-3096 ◽

Cited By ~ 16

Author(s):

E R Kaufman

Keyword(s):

Mammalian Cells ◽

Culture Medium ◽

Chemical Mutagen ◽

Genetic Studies ◽

Cells In Culture ◽

Thymidine Analog ◽

High Concentrations ◽

Hypoxanthine Guanine Phosphoribosyltransferase ◽

High Degree ◽

Spontaneous Mutants

Two protocols have been developed, both of which utilize the thymidine analog 5-bromodeoxyuridine (BrdUrd) to induce mutations in mammalian cells in culture (E. R. Kaufman and R. L. Davidson, Proc. Natl. Acad. Sci. USA 75:4982-4986, 1978; E. R. Kaufman, Mol. Cell. Biol. 4:2449-2454, 1984). The first protocol, termed incorporational (INC) mutagenesis, utilizes high concentrations of BrdUrd in the culture medium to generate a high intracellular ratio of BrdUTP/dCTP. The second protocol, termed replicational (REP) mutagenesis, entails the incorporation of BrdUrd into DNA under nonmutagenic conditions, the removal of all BrdUrd from the culture medium, and the subsequent replication of the bromouracil-containing DNA in the presence of high intracellular levels of dTTP and dGTP. Genetic studies using reversion analysis at the hypoxanthine-guanine phosphoribosyltransferase locus were used to determine whether the mechanisms of these two BrdUrd mutagenesis protocols had enough specificity to be distinguishable by their ability to revert various mutants. The results of these studies indicated that (i) mutants induced by INC mutagenesis were induced to revert only by REP mutagenesis and not by INC mutagenesis, (ii) mutants induced by REP mutagenesis were more efficiently reverted by INC mutagenesis than by REP mutagenesis, and (iii) both spontaneous mutants and mutants induced by the chemical mutagen ethyl methanesulfonate showed a high degree of specificity when tested for reversion by the BrdUrd mutagenesis protocols.

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Reversion analysis of mutations induced by 5-bromodeoxyuridine mutagenesis in mammalian cells

Molecular and Cellular Biology ◽

10.1128/mcb.5.11.3092-3096.1985 ◽

1985 ◽

Vol 5 (11) ◽

pp. 3092-3096

Author(s):

E R Kaufman

Keyword(s):

Mammalian Cells ◽

Culture Medium ◽

Chemical Mutagen ◽

Genetic Studies ◽

Cells In Culture ◽

Thymidine Analog ◽

High Concentrations ◽

Hypoxanthine Guanine Phosphoribosyltransferase ◽

High Degree ◽

Spontaneous Mutants

Two protocols have been developed, both of which utilize the thymidine analog 5-bromodeoxyuridine (BrdUrd) to induce mutations in mammalian cells in culture (E. R. Kaufman and R. L. Davidson, Proc. Natl. Acad. Sci. USA 75:4982-4986, 1978; E. R. Kaufman, Mol. Cell. Biol. 4:2449-2454, 1984). The first protocol, termed incorporational (INC) mutagenesis, utilizes high concentrations of BrdUrd in the culture medium to generate a high intracellular ratio of BrdUTP/dCTP. The second protocol, termed replicational (REP) mutagenesis, entails the incorporation of BrdUrd into DNA under nonmutagenic conditions, the removal of all BrdUrd from the culture medium, and the subsequent replication of the bromouracil-containing DNA in the presence of high intracellular levels of dTTP and dGTP. Genetic studies using reversion analysis at the hypoxanthine-guanine phosphoribosyltransferase locus were used to determine whether the mechanisms of these two BrdUrd mutagenesis protocols had enough specificity to be distinguishable by their ability to revert various mutants. The results of these studies indicated that (i) mutants induced by INC mutagenesis were induced to revert only by REP mutagenesis and not by INC mutagenesis, (ii) mutants induced by REP mutagenesis were more efficiently reverted by INC mutagenesis than by REP mutagenesis, and (iii) both spontaneous mutants and mutants induced by the chemical mutagen ethyl methanesulfonate showed a high degree of specificity when tested for reversion by the BrdUrd mutagenesis protocols.

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Threonine synthesis from homoserine as a selectable marker in mammalian cells

Biochemical Journal ◽

10.1042/bj2990637 ◽

1994 ◽

Vol 299 (3) ◽

pp. 637-644 ◽

Cited By ~ 7

Author(s):

W D Rees ◽

S D Grant ◽

S M Hay ◽

K M Saqib

Keyword(s):

Escherichia Coli ◽

Active Region ◽

Cell Lines ◽

Hela Cells ◽

Mammalian Cells ◽

Selectable Marker ◽

Threonine Synthase ◽

Homoserine Kinase ◽

Mouse Cells ◽

High Concentrations

The plasmid pSVthrBC expresses the Escherichia coli thrB (homoserine kinase) and thrC (threonine synthase) genes in mouse cells and enables them to synthesize threonine from homoserine. After transfection with pSVthrBC and culture in medium containing homoserine, only cells that have incorporated pSVthrBC survive. Homoserine at concentrations greater than 1 mM is toxic to mammalian cells. Mouse cells selected from medium containing 5 mM homoserine had incorporated 20-100 copies of the plasmid per cell and had homoserine kinase activities of 0.001-0.012 nmol/min per mg of protein per copy. Cells selected from medium containing 10 mM homoserine had incorporated one or two copies of the plasmid per cell and had homoserine kinase activities of 0.06-0.39 nmol/min per mg of protein per copy. By using high concentrations of homoserine, it is possible to use pSVthrBC to select and isolate cell lines that have one or two copies of the plasmid incorporated into an active region of chromatin. CHO and HeLa cells have also been successfully transfected with pSVthrBC. COS-7 cells are naturally resistant to homoserine as they are able to metabolize homoserine.

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