APRT: a versatile in vivo resident reporter of local mutation and loss of heterozygosity.

We describe an in vivo mutagenesis model that utilizes reverse mutation and forward mutation at the endogenous Aprt locus. Reverse mutation provides an in situ method for detecting environments or agents that cause point mutations. Forward mutation detects large chromosomal events, including mitotic recombination, chromosome loss, and large multilocus deletion, all of which can lead to loss of heterozygosity. Detection of reverse mutation in vivo is based on the differential capacity of Aprt and Aprt cells to sequester radiolabeled adenine by catalyzing its conversion to adenosine monophosphate with subsequent incorporation into nucleic acids. Cells lacking APRT activity cannot accumulate exogenously administered, tagged adenine, whereas Aprt+ cells can and will thereby become marked. Thus, genetically modified mice with mutant but revertible Aprt alleles should be a useful vehicle for in situ detection of mutagenic activity in the whole animal. the feasibility of this model has been illustrated, first, by showing that APRT-deficient mice are viable and, second, by demonstrating that the minority of Aprt+ cells within a chimeric tumor growing in an Aprt+ mouse can be selectively labeled following IP injection of [14C]-adenine and can be identified by autoradiography. Forward mutation, detected by growth in selective medium of primary cells derived from Aprt+/- heterozygous mice, provides on independent estimate of in vivo mutation frequency. The frequency with which Aprt colonies arise provides a measure of the frequency of Aprt(-)-negative cells in the tissue at that point in time. Culture of skin fibroblasts in 2,6-diaminopurine (DAP) produced Aprt+ colonies with a frequency of about 10(-4). This frequency is similar to that found for human T lymphocytes from individuals heterozygous at the Aprt locus. In both cases, the majority of mutagenic events involved allele loss. Polymerase chain reaction with linked polymorphic microsatellites on mouse chromosome 8 demonstrated that allele loss was mediated mostly by mitotic recombination, as was the case for human T lymphocytes. The high frequency of mitotic recombination and allele loss at a neutral locus has significant implications for the process of tumorigenesis and argues that spontaneous or induced mitotic recombination may play a causal role in the progression to cancer.