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 PhiX174 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 PhiX174 (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 approximately 10 times higher, for unknown reasons. 
We conclude that the spontaneous mutant frequencies measured by the lacI and 
PhiX174 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.