A method to quantify spontaneous and in vivo induced thioguanine-resistant mouse lymphocytes

Jones, Irene M.; Burkhart-Schultz, Karolyn; Carrano, A.V.

doi:10.1016/0165-1161(85)90022-6

Cited by 94 publications

(36 citation statements)

References 11 publications

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“…There is a question of the relevance of these in vitro mutant frequencies and spectra of mutation to those produced in vivo. In vivo models for mutation detection in the endogenous X-linked Hprt gene were developed for various animal species and man [Albertini et al, 1982;Jones et al, 1985;Aidoo et al, 1997]. Later, recoverable transgenes were introduced into the germline of rodents to serve as a target for mutagenesis [Kohler et al, 1991;Gossen et al, 1994].…”

Section: Introductionmentioning

confidence: 99%

Mutagenicity of γ‐radiation, mitomycin C, and etoposide in the Hprt and Tk genes of Tk^+/− mice†

Dobrovolsky

Shaddock

Heflich

2002

Environ and Mol Mutagen

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The recently developed Tk(+/-) mouse detects in vivo somatic cell mutation in the endogenous, autosomal Tk gene. To evaluate the sensitivity of this model, we have treated Tk(+/-) mice with three agents that induce DNA damage by different mechanisms, and determined spleen lymphocyte mutant frequencies (MFs) in the autosomal Tk gene and in the X-linked Hprt gene. gamma-Radiation, which produces single- and double-strand breaks by nonspecific oxidative stress, efficiently increased Hprt MF, but not Tk MF. Mitomycin C, which produces bulky DNA monoadducts and crosslinks, was mutagenic in both the Hprt and Tk genes, but the response was greater in the Tk gene. An inhibitor of the ligase function of DNA topoisomerase II, etoposide, did not increase Hprt MF, and induced a small, but nonsignificant increase in Tk MF. Combined with previous data, the results indicate that the two genes are differentially sensitive to many agents, and that the Tk gene is more sensitive than the Hprt gene to some, but not all types of DNA damage.

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Section: Introductionmentioning

confidence: 99%

Mutagenicity of γ‐radiation, mitomycin C, and etoposide in the Hprt and Tk genes of Tk^+/− mice†

Dobrovolsky

Shaddock

Heflich

2002

Environ and Mol Mutagen

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“…Unfortunately, assays involving endogenous genes are either insensitive, as in the case of polymerase chain reaction (PCR)-based assays, or limited to very particular tissues or developmental stages, as in the case of specific locus assays involving endogenous loci [Jones et al, 1985;Winton et al, 1988, Aidoo et al, 1991. Transgenic mutation assays created in mice and rats are not subject to these limitations [Gossen et al, 1989;Kohler et al, 1991;Burkhart et al, 1992, Boerrigter et al, 1995Manjanatha et al, 1998].…”

Section: Introductionmentioning

confidence: 99%

In vivo transgenic mutation assays

Heddle

Dean²,

Nohmi

et al. 2000

Environ. Mol. Mutagen.

117

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Transgenic rodent gene mutation models provide quick and statistically reliable assays for mutations in the DNA from any tissue. For regulatory applications, assays should be based on neutral genes, be generally available in several laboratories, and be readily transferable. Five or fewer repeated treatments are inadequate to conclude that a compound is negative but more than 90 daily treatments may risk complications. A sampling time of 35 days is suitable for most tissues and chemicals, while shorter sampling times might be appropriate for highly proliferative tissues. For phage‐based assays, 5 to 10 animals per group should be analyzed, assuming a spontaneous mutant frequency (MF) of ∼3 × 10−5 mutants/locus and 125,000–300,000 plaque or colony forming units (PFU or CFU) per tissue. Data should be generated for two dose groups but three should be treated, at the maximum tolerated dose (MTD), two‐thirds the MTD, and one‐third the MTD. Concurrent positive control animals are only necessary during validation, but positive control DNA must be included in each plating. Tissues should be processed and analyzed in a block design and the total number of PFUs or CFUs and the MF for each tissue and animal reported. Sequencing data would not normally be required but might provide useful additional information in specific circumstances. Statistical tests used should consider the animal as the experimental unit. Nonparametric statistical tests are recommended. A positive result is a statistically significant dose‐response and/or statistically significant increase in any dose group compared to concurrent negative controls using an appropriate statistical model. A negative result is statistically nonsignificant with all mean MF within two standard deviations of the control. Environ. Mol. Mutagen. 35:253–259, 2000 © 2000 Wiley‐Liss, Inc.

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“…Splenocytes were cultivated in general as described by Jones et al (1). In brief, spleen cells are isolated from 3 to 6 adult male C57bl mice and cultured in roundbottomed 96-well microtiter plates in the presence of 60 Gy irradiated feeder cells (104 hprt-L5178Y mouse lymphoma cells and a variable number of splenocytes).…”

Section: Thioguanine-resistant Mutants and Cultivation Of Splenocytesmentioning

confidence: 99%

“…Modified RPMI1640 medium in an atmosphere of 5% CO2 was used for culture. The medium was supplemented with 10% fetal calf serum, HEPES (25 mM), mercaptoethanol (5 x 10-5 mM), 0.2 mg/mL sodium pyruvate, recombinant IL-2 (100 U/mL)i penicillin (100 U/mL), streptomycin (100 ,ug/mL), 1 ,ug/mL indomethacin, and 5 jig/mL Con A. Medium of selective plates contained in addition 2.5 ,ug/mL 6-thioguanine (TG).…”

Section: Thioguanine-resistant Mutants and Cultivation Of Splenocytesmentioning

confidence: 99%

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Quantification of thioguanine-resistant lymphocytes from mice irradiated in vivo.

Lorenz

Gollner

Hempel

1990

Environ Health Perspect

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Adult mice were Co-60 gamma irradiated, and 7 months later splenocytes were isolated, cultured in microwells, and the frequency of hprt-deficient mutants was determined by measuring the cloning efficiency in media with 6-thioguanine. The mutant frequency at 2, 4, and 6 Gy was 1.6 x 10(-5), 4.4 x 10(-5), and 12.7 x 10(-5), respectively. The frequency of spontaneous mutants was 2.5 x 10(-6). The effect of metabolic cooperation on the cloning efficiency of thioguanine-resistant T-cells in selective medium was evaluated in co-cultures with wild-type T-cells. We found that the growth of hprt-deficient T-cells is supported in the presence of thioguanine-inactivated wild-type splenocytes up to a cell density of 5 x 10(5) cells per well. When cell density was higher, cell growth was inhibited. Possibilities and limitations of cloned lymphocytes for the analysis of somatic mutations that occur in vivo are discussed.

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A method to quantify spontaneous and in vivo induced thioguanine-resistant mouse lymphocytes

Cited by 94 publications

References 11 publications

Mutagenicity of γ‐radiation, mitomycin C, and etoposide in the Hprt and Tk genes of Tk^+/− mice†

Mutagenicity of γ‐radiation, mitomycin C, and etoposide in the Hprt and Tk genes of Tk^+/− mice†

In vivo transgenic mutation assays

Quantification of thioguanine-resistant lymphocytes from mice irradiated in vivo.

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A method to quantify spontaneous and in vivo induced thioguanine-resistant mouse lymphocytes

Cited by 94 publications

References 11 publications

Mutagenicity of γ‐radiation, mitomycin C, and etoposide in the Hprt and Tk genes of Tk+/− mice†

Mutagenicity of γ‐radiation, mitomycin C, and etoposide in the Hprt and Tk genes of Tk+/− mice†

In vivo transgenic mutation assays

Quantification of thioguanine-resistant lymphocytes from mice irradiated in vivo.

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Product

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Mutagenicity of γ‐radiation, mitomycin C, and etoposide in the Hprt and Tk genes of Tk^+/− mice†

Mutagenicity of γ‐radiation, mitomycin C, and etoposide in the Hprt and Tk genes of Tk^+/− mice†