Mouse spermatogonia exposed to a high, multiply fractionated dose of a cancer chemotherapeutic drug: Mutation analysis by electrophoresis

Johnson, F. M.; Lewis, Susan E.

doi:10.1016/0027-5107(81)90034-8

Cited by 15 publications

(2 citation statements)

References 6 publications

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“…A small control series, 10 animals from each strain, were injected with buffer only and mated in the same way. Additional control data were obtained from previous experiments (8,9).…”

Section: Methodsmentioning

confidence: 99%

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Electrophoretically detected germinal mutations induced in the mouse by ethylnitrosourea.

Johnson¹,

Lewis²

1981

Proc. Natl. Acad. Sci. U.S.A.

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Male DBA/2J and C57BL/6J mice were treated with ethylnitrosourea (EtNU) at a dose of 250 mg/kg of body weight. After a sterile period of 11 weeks, the treated animals were mated with untreated females; treated DBA with C57 and treated C57 with DBA. A small control group of untreated males was established, and a larger control group from earlier experiments was also used for comparison Electrophoretic analysis has demonstrated genetic alterations in a wide variety of organisms, and the approach is currently being used to screen for mutations in human populations (6, 7). This paper presents the results of the application of the electrophoretic approach to the detection ofgerminal mutations induced by EtNU. MATERIALS AND METHODSThe two strains of mice used-C57BL/6J and DBA/2J-were purchased from The Jackson Laboratory. Seventy DBA male mice were injected with a solution of EtNU and subsequently mated with C57 females. Likewise, 70 C57 males were injected and mated with DBA females. Only one death occurred among the treated males following treatment. A small control series, 10 animals from each strain, were injected with buffer only and mated in the same way. Additional control data were obtained from previous experiments (8, 9).The EtNU was obtained from Bio-Clinical Laboratories (Bohemia, NY). Before injection, it was dissolved in 0.05 M sodium phosphate buffer (pH 4.0) at 25 mg/ml. The solution, volume adjusted to deliver a dose of 250 mg/kg of body weight, was administered by intraperitoneal injection. All injections were completedwithin 10 min ofpreparation ofthe solution. Controls received only buffer. The first trial matings were attempted 5 or 6 weeks after injection; when the sterile period ended (10.5 + 1.0 weeks after injection), a regular schedule of mating was established according to which the males were each placed with a pair of females in mating cages for 1 week. At the end of that time, each male was placed with a different pair offemales. The schedule was maintained for several months to produce F1 animals. The offspring were weaned at m24 days of age and used for electrophoretic analysis when =-8-10 weeks old.The sample material for electrophoretic studies consisted of erythrocyte lysates and kidney homogenates. The samples were obtained surgically, so the animals were alive and available for mating to determine heritability ofthe detected variations. The parent animals and all of their F1 progeny were examined by the same set of techniques. Methods for sample preparation, electrophoresis, and gel staining have been described (8). The techniques allow genetic variation to be detected at 21 loci, which are of two types.For one set of 10 loci, there are electrophoretic differences between the strains determined by alternative homozygous alleles in the two strains. These loci are Mod-i, Es-i, Es-3, Idh-1, Hbb, Pep-3, Gpd-i, Gpi-i, Pgm-i, and Pgm-3. Loci of this type are especially useful because they make it possible to tell from which parent a newly arisen mutational event originated.The ot...

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“…A small control series, 10 animals from each strain, were injected with buffer only and mated in the same way. Additional control data were obtained from previous experiments (8,9).…”

Section: Methodsmentioning

confidence: 99%

“…In total, 290,252 control locus tests were performed in the present and two other experiments (8,9). No newly arisen spontaneous mutations were found in this group (see Table 1), but some mutations preexisting in the parental animals were identified (8).…”

mentioning

confidence: 99%

Electrophoretically detected germinal mutations induced in the mouse by ethylnitrosourea.

Johnson¹,

Lewis²

1981

Proc. Natl. Acad. Sci. U.S.A.

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The agar contact replica technique after isoelectric focusing as a screening method for the detection of enzyme variants

Pretsch¹,

Charles²,

Narayanan³

1982

Electrophoresis

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Electrophoresis 1982,3,[142][143][144][145] affected simply by adsorption to nitrocellulose). Further extensive studies will be necessary to determine the proportion of monoclonals directed against such tertiary and secondary structural antigens, but it seems likely to be high. Experiments in this laboratory attempting to use commercial monoclonal antibodies to identify human lymphocyte antigens (P2-microglobulin, HLA, etc.) or to stain several as yet unidentified human urinary proteins on two-dimensional gel transfers have so far been uniformly unsuccessful. Hence the use of monoclonal antibodies to stain spots on two-dimensional transfers may be considered problematical, unless the antibodies are initially selected for the ability to recognize an antigen in a denatured state.The results with animal antisera confirm a majority of the protein identifications made previously in the human plasma two-dimensional pattern [91, and have extended those results by providing six new identifications. An updated diagram (Figure 5 ) now shows a total of 37 polypeptides identified. Small amounts of specific antibody to protein spots not yet isolated biochemically may be obtained by ammonium thiocyanate elution of immunoglobulins from spots in a transfer stained with a polyspecific (i.e., anti-human serum) antiserum. Preliminary experiments suggest that such a procedure will be particularly valuable in preparing antibody to a variety of lymphoid cell proteins.Electrofocusing has been adaptea in order to make screening experiments tor tne detection of enzyme variants more efficient. The principle of the new technique consists in making agar copies of the polyacrylamide gel by contact; the original gel and the agar copies are then developed for different enzyme systems. By doing so, the number of enzymes, i. e. loci, per test animal screened after a single electrofocusing run is increased. This method has proved to be feasible for the detection of mouse variants with altered enzyme banding patterns due to parental treatment with chemical mutagens.

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The biochemical specific‐locus test and a new multiple‐endpoint mutation detection system: Considerations for genetic risk assessment

Lewis

1991

Environ and Mol Mutagen

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The importance of studying the generation of mutations in mouse germ cells is emphasized, and the utility of the biochemical specific-locus test in detecting germinal mutations is described. All experiments performed to date using this test are listed. The relevance of dominant mutations to human genetic risk is also discussed. Finally, the value of using multiple tests for the study of in vivo germinal mutations is discussed, and the design for a new multiple-endpoint system is presented.

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Mouse spermatogonia exposed to a high, multiply fractionated dose of a cancer chemotherapeutic drug: Mutation analysis by electrophoresis

Cited by 15 publications

References 6 publications

Electrophoretically detected germinal mutations induced in the mouse by ethylnitrosourea.

Electrophoretically detected germinal mutations induced in the mouse by ethylnitrosourea.

The agar contact replica technique after isoelectric focusing as a screening method for the detection of enzyme variants

The biochemical specific‐locus test and a new multiple‐endpoint mutation detection system: Considerations for genetic risk assessment

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