DETERMINATION OF SPONTANEOUS LOSS OF HETEROZYGOSITY MUTATIONS IN APRTHETEROZYGOUS 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 heterozygous and homozygous mice were derived. Aprt homozygous deficient animals were viable thoug h the mendelian inheritance pattern was skewed, On average these mice died at 6 months of age from severe renal failure. In T-lymphocytes of Apt heterozygous mice the mean spontaneous mutant frequency at the Ap rt 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 wh ether LOH events contribute to the high spontaneous mutant frequency a t the Aprt locus, 140 Aprt mutant T-lymphocyte clones were expanded an d analysed by allele-specific PCR, In 97 (69%) of these clones the wil d-type allele had been lost, Nine of the mutant clones were characteri zed in more detail using dual-coloured fluorescence in situ hybridizat ion 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 tar geted allele, Therefore, mitotic recombination or chromosome loss foll owed by duplication of the remaining homologue appears to be the predo minant mechanism for the in vivo generation of Aprt mutant T-lymphocyt es.