Determination of spontaneous loss of heterozygosity mutations in Aprt heterozygous mice

A mouse model was generated to investigate loss of heterozygosity (LOH) events in somatic cells. The adenine phosphoribosyltransferase ( Aprt ) gene was disrupted in embryonic stem cells using a conventional gene targeting approach and subsequently Aprt hetero-zygous and homozygous mice were derived. Aprt homozygous deficient animals were viable though the mendelian inheritance pattern was skewed. On average these mice died at 6 months of age from severe renal failure. In T-lymphocytes of Aprt heterozygous mice the mean spontaneous mutant frequency at the Aprt locus was 8.7 x 10(-6) while the frequency was 0.8 x 10(-6) at the hypoxanthine phosphoribosyltransferase locus. In order to determine whether LOH events contribute to the high spontaneous mutant frequency at the Aprt locus, 140 Aprt mutant T-lymphocyte clones were expanded and analysed by allele-specific PCR. In 97 (69%) of these clones the wild-type allele had been lost. Nine of the mutant clones were characterized in more detail using dual-coloured fluorescence in situ hybridization analysis. Five out of six of the mutant clones which arose from an LOH event, based on the PCR assay, contained a duplication of the targeted allele. Therefore, mitotic recombination or chromosome loss followed by duplication of the remaining homologue appears to be the predominant mechanism for the in vivo generation of Aprt mutant T-lymphocytes.