Spectra of spontaneous and mutagen-induced mutations in the lacI gene in transgenic mice.

Abstract

Transgenic nmice with a A shuttle vector containing a lacI target gene were generated for use as a shortterm, in vivo mutagenesis assay. The gene is recovered from the treated mice by exposing mouse genomic DNA to in vitro packaging extracts and plating the rescued phage on agar plates containing 5-bromo-4-chloro-3-indolyl (3-D-galactopyranoside (X-Gal). Phage with mutations in the lacl gene form blue plaques, whereas phage with a nonmutated lacl form colorless plaques. Spontaneous background mutant rates using this system range from 0.6 x 10-S to 1.7 x i0-0, depending upon tissue analyzed, with germ cells exhibiting less than one-third the background rate of somatic tissue. Treatment of the miceor cyclophosphamide caused an induction of mutations over background. Recovery of the lacI target for sequence analysis was performed by genetic excision of a plasmid from the phage using partial filamentous phage origins. The predominant mutations identified from untreated and treated populations were base substitutions. Although it has been shown by others that 70% of all spontaneous mutations within the laI gene, when replicated in Escherichia cohi, occur at a hot spot located at bases 620-632, only 1 of 21 spontaneous mutations has been identified in this region in the transgenic mouse system. In addition, 5 of 9 spontaneous transitions analyzed occur at CpG dinucleotides, whereas no transition mutations were identified at the prokaryotic deamination hot spots occurring at dcm sites (CCA/TGG) within the WaI gene. For EtNU, approximately equal amounts of transitions and transversions were observed, contrasting with B[a]P-induced mutations, in which only transversions were obtained. In addition, B[a]P mutagenesis showed a predominance ofmutations (81 %) involving cytosines and/or guanines, consistent with its known mode of action. The discovery of a spontaneous mutation spectrum different from that of bacterial assays, coupled with the concordance of EtNU and B[a]P base mutations with the known mechanisms of activity for these mutagens, suggests that this transgenic system will be useful as a short-term, in vivo system for mutagen assessment and analysis of mechanisms leading to mutations.The lacd gene has been used extensively as a target for the identification and analysis of spontaneous and induced mutations, due in part to the ease of using a calorimetric assay to rapidly screen for mutations. Genetic and sequence analysis of spontaneous and induced mutations detected in several systems utilizing lacd (1-5) has resulted in an extensive accumulation of data regarding the sequence specificity of spontaneous and induced mutations in lacd, which can be of considerable comparative value through the use of the lacd target gene in whole animal assay systems.To combine the cost-saving aspects of short-term assays with the predictive power of whole animal assays, we previously described the development of a system that depends upon efficient recovery of a A phage shuttle vector from transgenic mouse genomic DNA thro...

Mice over-expressing human O6alkylguanine-DNA alkyltransferase selectively reduce O6methylguanine mediated carcinogenic mutations to threshold levels after N-methyl-N-nitrosourea

“…Mutation frequencies represent (a) pooled data from the six Big Blue TM /hMGMT+ and seven Big Blue TM mice and expressed as a mutation frequency per cohort by tissue (mutations/PFU, strati®ed by tissue) (Table 3), and (b) the mean mutation frequency per mouse by tissue (mutations per mouse/PFU per mouse) ( Figure 2), measured in each of the three tissues for the two mouse cohorts. Background mutation frequency was very similar to that previously reported (Kohler et al, 1991;de Boer et al, 1998). MNU is a very powerful mutagen, increasing the mutation frequency 32-fold over background in the thymus and 13-fold in the spleen of Big Blue TM mice.…”

“…Table 2 shows that a G : C to A : T mutation in the second position of codon 12 (GGT to GAT) was present in 43 out of 59 lymphomas from mice lacking the hMGMT transgene; 38% of lymphomas from mice treated with 50 mg/kg MNU, 53% from mice treated at 80 mg/kg MNU and 69% from mice treated with The alkyltransferase assay has been published in detail (Liu et al, 1994). Brie¯y, activity was measured in sonicated tissue extracts by quantifying the amount of (Dumenco et al, 1993) were bred to heterozygous male Big Blue TM (lacI+/B6; Stratagene, La Jolla, CA, USA), (Kohler et al, 1991) mice. O spring were treated with 80 mg/kg MNU at 6 weeks of age and followed for 1 year.…”

Direct mutation analysis by high-throughput sequencing: From germline to low-abundant, somatic variants

“…The need for an in vivo assay that can be applied to all organs and tissues led to the development of transgenic reporter based methods. Mice harboring a LacZ or LacI reporter transgene emerged in the late 80s [48–50]. These reporter genes are part of a lambda or plasmid construct that can be recovered from genomic DNA and tested in E. coli for mutational inactivation of the reporter gene.…”

Recovery of a low mutant frequency after ionizing radiation-induced mutagenesis during spermatogenesis

“…Extrapolation from animal data has been used in an effort to understand IRinduced germline mutations in humans due to the inherent difficulties in human epidemiological studies involving germ cells [1]. Several rodent assays are capable of detecting germline mutation, including the specific locus (SL) test [2], heritable translocation (HT) assay [3], dominant lethal (DL) assay [4], expanded simple tandem repeats (ESTRs) assay [5], and transgenic rodent assays [6,7]. The traditional mutation assays, e.g.…”

“…In a Lac I transgenic mouse system, the bacteriophage λ genome carrying the Lac I repressor gene from the prokaryotic Lac operon was introduced into the mouse genome [7]. The λ DNA is recovered from mouse genomic DNA and used to infect Escherichia coli carrying a Lac Z (β-galactosidase) gene (ΔLacZ M15), but lacking a functional Lac I gene.…”