Comparison of mutant frequencies and types of mutations induced by thiotepa in the endogenous Hprt gene and transgenic lad gene of Big Blue® rats

The utility of the lad transgene of Big Blue(R) rats as a reporter of in vivo mutation was evaluated by comparing the frequency and types of mutations induced by thiotepa in the transgene and the endogenous Hprt gene. Transgenic rats were given i.p. injections of 1.4 mg/kg of thiotepa three times per week over a period of 4 weeks (a total dose of 16.8 mg/kg); 1 week after the last injection, mutation assays were performed on spleen lymphocytes isolated from the animals. Thiotepa treatment increased the lad mutant frequency from 34.8 +/- 4.1 x 10(-6) in control animals to 140.9 +/- 64.8 x 10-6 (p = 0.0020) and the Hprt mutant frequency from 3.5 +/- 1.5 x 10-6 to 41.1 +/- 23.2 x 10-6 (p = 0.0028). Sequence analysis of lad mutant DNA and Hprt mutant cDNA produced similar overall mutation patterns: G:C --> T:A transversion was the most common base pair substitution (32% of independent mutations in the lad gene and 28% of Hprt mutations), and deletions and insertions accounted for 34% of mutations in the lacI gene and 28% in the Hprt gene. The majority of thiotepa-induced base pair substitutions in the Hprt gene occurred with the mutated purine on the non-transcribed DNA strand, while no strand-related bias was found for mutations in the lad gene. Substitutions at G:C base pairs in the lad gene, but not in the Hprt gene, were found disproportionately in CpG sites. In addition, multiplex polymerase chain reaction analysis of genomic DNA from the Hprt mutants indicated that 34% had relatively large deletions; none of these deletions was detected by the cDNA analysis. The results indicate that the frequency of thiotepa-induced mutants in Big Blue(R) rats was 2.8-fold greater in the lad gene than in the Hprt gene. Although the Hprt gene recovered a fraction of large deletions not found among the lad mutants, the effects of transcription-coupled DNA repair in the Hprt gene and the targeting of base pair substitutions to G:C base pairs in CpG sites may have contributed to the higher mutant frequencies induced by thiotepa in the lad transgene compared with the Hprt gene. (C) 1998 Elsevier Science B.V. All rights reserved.