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The ΦX174 transgenic mouse was first developed as an in vivo Ames test, detecting base pair substitution (bps) at a single bp in a reversion assay. A forward mutational assay was also developed, which is a gain of function assay that also detects bps exclusively. Later work with both assays focused on establishing that a mutation was fixed in vivo using single-burst analysis: determining the number of mutant progeny virus from an electroporated cell by dividing the culture into aliquots before scoring mutants. We review results obtained from single-burst analysis, including testing the hypothesis that high mutant frequencies (MFs) of G:C to A:T mutation recovered by transgenic targets include significant numbers of unrepaired G:T mismatches. Comparison between the ΦX174 and lacI transgenes in mouse spleen indicates that the spontaneous bps mutation frequency per nucleotide (mf(n)) is not significantly lower for ΦX174 than for lacI; the response to ENU is also comparable. For the lacI transgene, the spontaneous bps mf(n) is highly age-dependent up to 12 weeks of age and the linear trend extrapolates at conception to a frequency close to the human bps mf(n) per generation of 1.7 × 10(-8). Unexpectedly, we found that the lacI somatic (spleen) bps mf(n) per cell division at early ages was estimated to be the same as for the human germ-line. The bps mf(n) in bone marrow for the gpt transgene is comparable to spleen for the lacI and ΦX174 transgenes. We conclude that the G:C to A:T transition is characteristic of spontaneous in vivo mutation and that the MFs measured in these transgenes at early ages reflect the expected accumulation of in vivo mutation typical of endogenous mammalian mutation rates. However, spontaneous and induced mf(n)s per nucleotide for the cII gene in spleen are 5-10 times higher than for these other transgenes., (Published by Elsevier B.V.)

The use of transgenic targets for measuring mutant frequencies in mammalian tissue requires an estimate of the mutant frequency that results from recovery of the transgene in bacterial recovery systems. In this study, we have determined the spontaneous mutant frequency, estimated the mutation rate, and ascertained the mutation spectrum for gene A of phiX174 grown in E. coli strain CQ2 from 156 small independent cultures. The mutant frequency of 12 of the 156 cultures was 17 +/- 1.0 x 10(-6) and the estimated mutation rate per gene replication was 7.4 +/- 2.3 x 10(-6). The mutant frequency and spectrum from E. coli were not significantly different from that of solvent-treated embryonic mouse cells in culture, 19 +/- 0.5 x 10(-6) (Valentine CR et al. [2002]: Environ Mol Mutagen 39:55-68), indicating that those spontaneous mutants were primarily derived from E. coli. The E. coli spectrum was heavily weighted toward two major target sites (hot spots), 4225A-->G (56%) and 4218G-->A or C (20%). Four new target sites and one new mutational event were recovered by the gene A forward assay. A mutant spectrum from an expanded phage stock was also determined to assess the effects of propagating the virus. This mutant frequency was higher (6 x 10(-4)), contained more double mutants (15% compared to 0.6%), and had a significantly different spectrum from the spectrum for independent cultures (fewer A:T-->G:C and G:C-->C:G changes and more G:C-->A:T; P < 0.002). The E. coli mutation spectrum will be useful for determining the origin of gene A mutation in tissues of phiX174 transgenic mice., (Copyright 2004 Wiley-Liss, Inc.)

Single-burst analysis was applied to a forward assay for gene A mutation in splenic lymphocytes of phiX174 transgenic mice for the purpose of optimizing analytical parameters for identifying in vivo mutations. The effect of varying the cutoff value for an in vivo burst on induced mutant frequency, fold increase, and the significance of the difference between control and N-ethyl-N-nitrosourea (ENU)-treated mice was calculated by two different methods. The plating density was reduced to an average of less than 10 background mutant plaques per aliquot in order to separate in vitro bursts. The spectrum of mutations contributing < 60 plaques per aliquot from control animals was not significantly different from the control spectra from E. coli or transgenic phiX174 cells in culture. The mutant spectra from ENU-treated animals was highly different between mutant bursts of > 80 plaques per aliquot compared to mutations contributing < 60 plaques per aliquot (P < 0.000001), the former fitting the spectrum expected for ENU-induced mutations. The latter spectrum was also different from control animals and E. coli (P < 0.000001), suggesting the difference was caused by ex vivo mutation. With the mutations found in this study, the total number of reported target sites for gene A is now 33. The results support the interpretation that, in contrast to results for the lacI transgene, 100% of mutants isolated in gene A from control animals and cells were fixed in E. coli. We attribute the difference between the two genes to hot-spot sites for mutation in gene A and to a testable hypothesis that the mosaic plaque assay for the lacI transgene underestimates the frequency of ex vivo mutants., (Copyright 2004 Wiley-Liss, Inc.)

Transgenic systems for measuring mammalian mutagenesis often use recoverable viral vectors. We hypothesize that mutations in these transgenic systems can arise from three different origins of DNA damage and replication errors and that these three origins of mutations (in vivo, ex vivo, and in vitro) can be differentiated in the PhiX174 am3, cs70 single burst assay (SBA) on the basis of burst size (BS). In vivo mutations are fixed in the animal, ex vivo mutations are fixed in bacterial cells during recovery of the phage, and in vitro revertants arise during the first replications of nonmutant phages under selective conditions. PX-2 cells, derived from a homozygous embryo of a PhiX174 transgenic mouse, were treated with vehicle or N-ethyl-N-nitrosourea (ENU). An algorithm was developed to estimate the BS that resulted in the highest induced revertant frequency; the estimate was 56. In vivo revertants were defined as having BS >55, ex vivo revertants as having a BS of 13-56, and in vitro revertants as having a BS of <14. The frequencies of in vivo revertants at 0, 100, and 200 mg/kg ENU were 0.06, 0.36, and 4.10 x 10(-6) (dose response, P = 0.004); ex vivo revertants were 0.36, 0.46, and 0.41 x 10(-6) (P = 0.37), and in vitro revertants were 0.39, 0.46, and 0.41 x 10(-6) (P = 0.55), respectively. These results show that only in vivo revertants reflect mutagen treatment. They also provide a basis for identifying PhiX174 am3 revertants induced in vivo and may increase the sensitivity of the assay for in vivo mutation.

Target genes in most transgenic systems have higher spontaneous mutation frequencies than do endogenous mammalian genes. Spontaneous mutations in transgenes predominantly arise from three sources: (1) mutations fixed in the animals, (2) mutations arising from replication errors caused by damage to the DNA that may have occurred in vivo or in vitro and then was fixed during amplification of the vector in vitro, and (3) mutations arising during replication of non-revertant phages in non-permissive bacteria. An assay based on single bursts was developed to directly distinguish between the in vivo and in vitro origins of revertants. The size of the aliquot is determined by mutant frequency and is adjusted so that ideally no more than 10 to 20% of the aliquots contain a bacterial cell transformed with a mutant phage. Mutations are detected as revertants of an amber mutation (am3) in phiX174 am3, cs70. The minimum burst size of non-revertant phiX am3, cs70 from splenic DNA on a permissive bacterial strain was larger than 30 plaque-forming units (pfu). Based on this observation, a burst size of 31 plaque-forming revertants was chosen as the minimum burst size of a fixed mutation. The single burst assay was tested on DNA from spleens of animals that were treated with 150 mg/kg 1-ethyl-1 nitrosurea. Only the fraction of aliquots with single bursts of revertants (> 30) increased in the treated animals compared to the controls. In contrast, there was no difference between treated and control animals for revertant frequencies calculated for burst sizes < or =30 pfu. Among the spontaneous mutations, only 30% were caused by mutations fixed in animals (i.e. burst size >30 pfu). Total average revertant frequency measured in DNA from treated animals was less than twofold more than the average spontaneous frequency (P = 0.048). When frequencies were based on burst sizes >30, there was a 4.6-fold increase among treated animals compared with controls (P = 0.026). The single burst-assay resulted in a more sensitive test for mutagenicity because it eliminated noise from in-vitro mutations.

In mutation assays using transgenic mice, with recoverable vectors such as PhiX174 am3, cs70, mutations originate from two sources: (1) in vivo mutations, that is, mutations that were fixed in the mouse, or (2) ex vivo mutations, that is, mutations that were fixed during recovery or plating. When a bacteriophage infects a bacterium, it multiplies and bursts the cell, releasing a number of phages referred to as the burst size. Our method for distinguishing between in vivo mutations and ex vivo mutations estimates the average number of bursts, the denominator of in vivo mutant frequencies, by dividing the total plaque-forming units (PFU) by the average number of phages in a burst. Herein, we outline a probability model relating observed plaque counts to the burst size and present the statistical method used to estimate the burst size. The average size of a single burst from nonrevertant phages was estimated in eight studies under the conditions of our mutation assay. The average burst size was stable across studies at 182.5 plaques per burst (standard error, 14.25). The probability that a burst is a specific size was approximated by a negative binomial distribution, which implies a Poisson-Pascal distribution for the observed plaque counts. The observed plaque counts were adequately fit by this approximation. Environ. Mol. Mutagen. 37:356-360, 2001 Published 2001 Wiley-Liss, Inc.