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

Abstract

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...

Genomic integration of lambda EG10 transgene in gpt delta transgenic rodents

“…In gpt mutation assays using gpt delta rats, we have frequently observed an A:T to T:A transversion at position 299 in the gpt sequence (a T t to a A t; Ile to Asn) (Additional file 5 : Fig. S5) [ 21 , 22 ]. The rescued gpt genes from F344 gpt delta rats without selection by 6TG were sequenced, and 2 of the 30 sequenced clones carried this mutation (data not shown).…”

“…In gpt delta rats, we have frequently observed an A:T to T:A transversion at position 299 in the gpt sequence [ 22 ]. The transgenes may contain inherited mutations in their sequences.…”

“…Because of this evidence, the A:T to T:A transversion at 299 in gpt is an inherited mutation of gpt delta rats that should be excluded from the mutation spectra. Interestingly, it was previously reported that 20 % of gpt mutants recovered from gpt delta rats contain this base substitution and speculated that one of the five copies may have the A:T to T:A mutation at 299 [ 22 ]. Therefore, we concluded that this gpt mutation must have arisen in one lambda EG10 copy in the development of gpt delta rats.…”

Prenatal exposure of mice to the human liver carcinogen aflatoxin B1 reveals a critical window of susceptibility to genetic change

“…The gpt transgene present in the recoverable phage lambda vector is not expressed in the mouse and is assumed to be genetically neutral, providing no advantage or disadvantage to cell growth. 36 Consequently, it would be expected that once mutations are fixed by DNA replication, the average MF in the liver would remain constant. However, liver cell organization and function are continuously being remodeled and reprogrammed, respectively, during the late fetal and early neonatal periods resulting in unequal growth of liver cell types that could increase or decrease the observed MF.…”

Oncomutations as biomarkers of cancer risk

“…A low frequency of spontaneous mutation was expected for single basepair targets based on neutral reporter gene measurements [McKinzie et al, 2001;Zhang et al, 2001;Lambert et al, 2005;Masumura and Nohmi, 2009] 27 . Using a random mutation capture assay with a sensitivity of 10 29 , Bielas and Loeb [2005a, b] reported a SMF per basepair of 1.6 3 10 28 in cultured human fibroblasts and a SMF <10 28 for several human tissues.…”