Development of a short-term,<i>in vivo</i>mutagenesis assay: the effects of methylation on the recovery of a lambda phage shuttle vector from transgenic mice

Kohler, S W; Provost, Gabrielle S. Le; Kretz, Patricia L.; Dycaico, Mark J.; Sorge, Joseph A.; Short, Jay M.

doi:10.1093/nar/18.10.3007

Cited by 120 publications

(40 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The establishment of transgenic mouse models for the quantitative detection of gene mutations (1,2) has provided unprecedented access to the study of mutagenesis in diverse tissues (3)(4)(5) and provided the basis for the establishment of a gene mutation assay in germ cells that would predict the transmission of induced mutations to subsequent generations. The process of accepting a transgenic mouse model for the detection and enumeration of induced gene mutations must involve the determination that the mutant frequencies (4,6) and the molecular characteristics of the mutations detected are representative of endogenous genes (6).…”

mentioning

confidence: 99%

Temporal and molecular characteristics of mutations induced by ethylnitrosourea in germ cells isolated from seminiferous tubules and in spermatozoa of lacZ transgenic mice.

Douglas

Jiao

Gingerich

et al. 1995

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

View full text Add to dashboard Cite

The lacZ transgenic mouse (Muta mouse) model was used to examine the timing of ethylnitrosourea (ENU)-induced mutations in germ cells. The spectrum of mutations was also determined. Animals received five daily treatments with ENU at 50 mg/kg and were sampled at times up to 55 days after treatment. In mixed germ-cell populations isolated from seminiferous tubules, there was little increase in the mutant frequency 5 days after treatment; subsequently, there was a continuous increase until the maximum (17.5-fold above background) was reached by -35 days. In the spermatozoa, an increase in mutant frequency was not seen until 20 days after treatment, with the maximum (4.3-fold above background) being achieved no sooner than -35 days. Based on the timing of sampling, these data demonstrate the detection of both spermatogonial and postspermatogonial mutations. The most prominent feature of the ENU-induced basepair mutations in testicular germ cells sampled 55 days after treatment is that 70%o are induced in AT base pairs, compared to only 16% in spontaneous mutations. These findings are consistent with comparable data from ENU studies using assays for inherited germ-cell mutations in mice. This study has demonstrated the utility and potential of the transgenic mouse lacZ model (Muta mouse) for the detection and study of germ-cell mutations and provides guidance in the selection of simplified treatment and sampling protocols.in bacteria, A-T base pairs are the predominate source of mutations in mammalian species in vivo (13). This change is related to the reduced capacity of DNA alkyltransferase to remove ethyl adducts other than 06-ethylguanine from mammalian cells (13). ENU induces both spermatogonial stem cell and postspermatogonial mutations in mice (14). The former are characterized by base-substitution mutations in A-T base pairs (15-19); the latter have been shown to result in relatively more multilocus lesions (20) and, if adducts are induced late in development, mosaic mutations in the progeny result (21).In this study we have used a lacZ transgenic mouse (Muta mouse) model to determine the timing of ENU-induced mutation induction in a mixed population of germ cells isolated from seminiferous tubules and in spermatozoa isolated from the vas deferens. The mutation spectrum for mutations from mixed germ cells was also determined. It is anticipated that such information will be influential in determining the acceptability of transgenic mouse models for the study of germ-cell mutagenicity, in suggesting appropriate experimental protocols for future studies, and in alleviating the need to conduct costly experiments on the offspring of exposed animals when it is not necessary. It is envisaged that such a transgenic mouse germ-cell assay, once validated, may become a companion to the dominant lethal test in terms of its role in hazard identification.The establishment of transgenic mouse models for the quantitative detection of gene mutations (1, 2) has provided unprecedented access to the study of mutagenesis ...

show abstract

mentioning

confidence: 99%

Temporal and molecular characteristics of mutations induced by ethylnitrosourea in germ cells isolated from seminiferous tubules and in spermatozoa of lacZ transgenic mice.

Douglas

Jiao

Gingerich

et al. 1995

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

View full text Add to dashboard Cite

show abstract

“…lacZ, lacI or cII have been employed as reporter genes in transgenic rodents, such as the Muta TM mouse, and the Big Blue R mouse and rat. [3][4][5][6][7] Despite differences in size and sequence context, spontaneous mutation frequencies of these reporter genes are in the mid-10 −5 range and those are predominantly base substitutions in most tissues. This high background of base substitutions may make it difficult to detect rare mutations such as deletions induced by ionizing radiation.…”

Section: Overview Of Gpt Delta Transgenic Rodentsmentioning

confidence: 99%

Spontaneous Mutagenesis in Rodents: Spontaneous Gene Mutations Identified by Neutral Reporter Genes in gpt Delta Transgenic Mice and Rats

Masumura

Nohmi

2009

JOURNAL OF HEALTH SCIENCE

View full text Add to dashboard Cite

Transgenic rodents are valuable models for investigation of genotoxicity of chemicals in vivo. We have developed gpt delta transgenic mice (C57BL/6J background) and rats (Sprague-Dawley, SD), which have the ability to identify both point mutations by the gpt assay [6-thioguanine (6-TG) selection] and certain types of deletions by the Spi − (Spi, sensitive to P2 interference) assay. Recently, the gpt delta SD rat was backcrossed with the Fisher 344 (F344) rat to establish an gpt delta F344 rat. The average spontaneous gpt mutation frequencies (MFs) are about 4.5 × 10 −6 in both SD and F344 gpt delta rats as well as in gpt delta mice. The G:C to A:T transitions at 5 -CpG-3 sites and G:C to T:A transversions are the predominant spontaneous gpt mutations in rats and mice. However, there is one false mutation (e.g. A:T to T:A at position 299) in the rats. The base substitution may have arisen when the lambda EG10 transgene was introduced into the genome of the SD rat during transgenesis. In the Spi − assay, 1-bp deletions in repetitive sequences are predominantly observed in both mice and rats. Possible mechanisms underlying the spontaneous mutations in gpt delta rodents are discussed.Key words --gpt delta transgenic rodent, spontaneous mutation, mutation spectrum, gpt assay, Spi − assay OVERVIEW OF gpt DELTA TRANSGENIC RODENTSGene mutations play an important role in the etiology of many human diseases including cancer. Since humans are exposed to a variety of endogenous and exogenous mutagens, there has been considerable interest in the relationship between exposure, genotoxic effects, and cancer incidence. To assess the risk of mutagens to the human genome, genotoxicity tests have been developed, including in vivo mutation assays using experimental animals, which play a crucial role in risk assessment. To investigate in vivo genotoxicity, a number of transgenic rodent mutation assays have been developed by introducing reporter transgenes into the chromosome of every cell of the animal. 1, 2) Using these systems, mutagenic events induced in a rodent can * To whom correspondence should be addressed: Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan. Tel.: +81-3-3700-1141 (Ext. 280); Fax: +81-3-3707-6950; E-mail: masumura@nihs.go.jp be detected by recovering the transgene and analyzing the phenotype of the reporter gene in a bacterial host. These models permit quantitation of mutations and identification at the sequence level in any tissue or organ in the body. lacZ, lacI or cII have been employed as reporter genes in transgenic rodents, such as the Muta TM mouse, and the Big Blue R mouse and rat. 3-7) Despite differences in size and sequence context, spontaneous mutation frequencies of these reporter genes are in the mid-10 −5 range and those are predominantly base substitutions in most tissues. This high background of base substitutions may make it difficult to detect rare mutations such as deletions induced by ionizing radiation. 8,9) To...

show abstract

mentioning

confidence: 99%

“…It has also been shown that some of these activities inhibit the transfer of lambda shuttle vector DNA from transgenic mice to E. coli cells (14,20,21,45). These inhibitory effects on lambda phage rescue result at least partially from the high levels of eukaryotic cytosine methylation occurring within the DNA (21). We had previously developed restriction-free lambda packaging extracts and plating strains in order to remove the inhibitory effect and increase the efficiency of these processes (21,22,23).…”

mentioning

confidence: 99%

“…These inhibitory effects on lambda phage rescue result at least partially from the high levels of eukaryotic cytosine methylation occurring within the DNA (21). We had previously developed restriction-free lambda packaging extracts and plating strains in order to remove the inhibitory effect and increase the efficiency of these processes (21,22,23). During the development of these extracts and strains, it was observed that strains containing the mcrA mcrBl genotype did not rescue methylated lambda DNA genomes as efficiently as did E. coli C strains.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Identification and characterization of a gene responsible for inhibiting propagation of methylated DNA sequences in mcrA mcrB1 Escherichia coli strains

Kretz¹,

Kohler²,

Short³

1991

J Bacteriol

View full text Add to dashboard Cite

Identifying and eliminating endogenous bacterial enzyme systems can significantly increase the efficiency of propagation of eukaryotic DNA in Escherichia coli. We have recently examined one such system which inhibits the propagation of lambda DNA rescued from transgenic mouse tissues. This rescue procedure utilizes lambda packaging extracts for excision of the lambda DNA from the transgenic mouse genome, as well as E. coli cells for subsequent infection and propagation. This assay, in combination with conjugal mating, P1 transduction, and gene cloning, was used to identify and characterize the E. coli locus responsible for this difference in efficiency. It was determined that the E. coli K-12 mcrB gene when expressed on a high-copy-number plasmid can cause a decrease in rescue efficiency despite the presence of the mcrBl mutation, which inactivates the classic McrB restriction activity. (This mutation was verified by sequence analysis.) However, this McrBl activity is not observed when the cloned mcrBl gene is inserted into the E. coil genome at one copy per chromosome. A second locus was identified which causes a decrease in rescue efficiency both when expressed on a high-copy-number plasmid and when inserted into the genome. The data presented here suggest that this locus is mrr and that the mrr gene product can recognize and restrict cytosine-methylated sequences. Removal of this DNA region including the mrr gene from E. coli K-12 strains allows high rescue efficiencies equal to those of E. coli C strains. These modified E. coli K-12 plating strains and lambda packaging extract strains should also allow a significant improvement in the efficiency and representation of eukaryotic genomic and cDNA libraries.

show abstract

Development of a short-term,in vivomutagenesis assay: the effects of methylation on the recovery of a lambda phage shuttle vector from transgenic mice

Cited by 120 publications

References 21 publications

Temporal and molecular characteristics of mutations induced by ethylnitrosourea in germ cells isolated from seminiferous tubules and in spermatozoa of lacZ transgenic mice.

Temporal and molecular characteristics of mutations induced by ethylnitrosourea in germ cells isolated from seminiferous tubules and in spermatozoa of lacZ transgenic mice.

Spontaneous Mutagenesis in Rodents: Spontaneous Gene Mutations Identified by Neutral Reporter Genes in gpt Delta Transgenic Mice and Rats

Identification and characterization of a gene responsible for inhibiting propagation of methylated DNA sequences in mcrA mcrB1 Escherichia coli strains

Contact Info

Product

Resources

About