Mutagenesis Advance Access published online on May 25, 2008
Mutagenesis, doi:10.1093/mutage/gen026
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Frequency and spectrum of ENU-induced mutation in the 
X174 transgene in mouse splenic lymphocytes and their significance to spontaneous transgenic rodent mutation frequencies
1Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, 3900 NCTR Road, HFT-120, Jefferson, AR 72079, USA 2Department of Epidemiology, College of Public Health, University of Arkansas for Medical, 4301 West Markham, #820 Little Rock, AR 72205, USA 3Present address: Cincinnati Children's Hospital Medical Center, Division of Allergy and Immunology, Cincinnati, OH 45040, USA 4Present address: University of Arkansas for Medical Sciences, School of Medicine, Little Rock, AR 72205, USA
A perceived disadvantage of transgenic rodent mutation assays is that spontaneous mutant frequencies are high compared to those of endogenous genes and may consequently reduce sensitivity to induced mutation. We have previously argued that unrepaired G:T mismatches from spontaneous deamination of 5-methylcytosine at CpG sites could be converted to apparent in vivo mutations in the bacterial recovery systems because of rapid, random, mismatch repair in Escherichia coli. In this study, we have measured mutation frequencies in spleen of male mice induced by N-ethyl-N-nitrosourea (ENU) using the 
X174 transgene, which is not subject to mismatch repair in E.coli, using single-burst analysis, a unique method to identify in vivo mutation. In order to compare our results to those using the lacI and cII transgenes, we converted all mutant frequencies to base pair substitution (bps) mutation frequencies per nucleotide based on mutant spectra from this study and published literature. We found this frequency in control spleen to be similar for lacI (3.8 ± 0.7 x 10–8) and X174 (3.1 ± 1.2 x 10–8) at 6 weeks of age. We found a strong age dependence for spontaneous lacI mutation that extrapolated to a value at conception (1.8 ± 0.9 x 10–8) that was not significantly different from the human germ line bps mutation frequency per nucleotide of 1.7 ± 0.2 x 10–8. These two transgenes provided similar mutational responses to 40 mg/kg ENU, 7- to 9-fold. In contrast, the cII target gene in the same tissue produces both spontaneous and induced mutation frequencies 
10 times higher, for unknown reasons. We conclude that the spontaneous mutant frequencies measured by the lacI and X174 transgenes in this moderately dividing tissue accurately measure in vivo mutation frequencies at early ages. For these two transgenes, seemingly high mutant frequencies may reflect the expected accumulation of somatic mutation with age.
* To whom correspondence should be addressed: Tel: +1 501 686 8849; Fax: +1 501 686 5845; Email: rdelongchamp{at}uams.edu
Received on February 20, 2008; revised on April 18, 2008; accepted on April 21, 2008.