FREQUENCY AND SPECTRUM OF ETHYLNITROSOUREA-INDUCED MUTATION AT THE HPRT AND LACI LOCI IN SPLENIC LYMPHOCYTES OF EXPOSED LACI TRANSGENIC MICE

The development of mouse models with the endogenous hypoxanthine-guani ne phosphoribosyl transferase (hprt) gene and lacI transgene as mutati onal targets provides an excellent opportunity to compare the mutant f requency (Mf) and types of mutations induced in vivo in different sequ ence contexts. To this end, a study was conducted to determine the Mfs and spectrum of mutations induced at these loci in splenic T cells fr om male B6C3F1 Big Blue mice (6 weeks old) exposed to N-ethyl-N-nitros ourea (ENU). Six weeks after i.p. injection of 40 mg ENU/kg, T cells w ere isolated from control (n = 7) and treated (n = 8) mice for the cul ture of hprt mutants and for the extraction of DNA and recovery of lac I mutants. Mutations in hprt exon 3 and in lacI were quantified and an alyzed using published procedures (S. W. Kohler et al., Proc. Natl. Ac ad. Sci. USA, 88: 7958-7962, 1991; T. R. Skopek et al., Proc. Natl. Ac ad. Sci. USA, 89: 7866-7870, 1992). In treated mice, the Mfs (average +/- SE) in hprt (6.0 +/- 0.2 x 10(-5)) and lacI (11.4 +/- 1.8 x 10(-5) ) were approximately 16.2-fold (P = 0.096) and 3.4-fold (P = 0.009), r espectively, above controls. However, the average induced Mfs (i.e., i nduced Mf = treatment Mf - background Mf) in hprt and lacI were simila r, with the respective increases in Mf being 5.6 +/- 0.2 x 10(-5) and 8.0 +/- 2.3 x 10(-5) over background. Eleven of the 107 hprt mutants f rom treated Big Blue mice had mutations in exon 3, with 73% being subs titutions at A . T bp. These data are similar to those observed in ENU -exposed nontransgenic B6C3F1 mice, in which 62 of 69 exon 3 mutations were substitutions at A . T bp (T. R. Skopek et al., Proc. Natl. Acad . Sci. USA, 89: 7866-7870, 1992). For comparison, the sequences of the lacI genes in two to five mutants from each mouse were determined, an d a total of 75 mutations (70 different mutations) was detected. In ex posed mice, 55% (24 of 44) of the mutations in lacI were substitutions at A . T bp. In controls, substitutions at AT bp comprised only 20% o f the recovered mutations in eithe hprt exon 3 (1 of 5) or lacI (5 of 26). These data indicate that the lacI mutation assay is less sensitiv e than the hprt assay for detecting increases in Mf induced by ENU exp osure of mice as indicated by the lower relative increase in Mf in the lacI gene, but, given a 6-week expression time, the types of mutation s induced by ENU in the transgene reflect those observed in the native transcribed gene.