Current models used to study the role of somatic mutation in aging have multiple drawbacks that limit their use in determining both types of mutations and the rate at which they accumulate in vivo. This project will validate a novel transgenic model for somatic mutation testing in aging research that, unlike current models, can be used for the quantitation and characterization of virtually all mutational types in any tissue. This validation will include the comparison of a novel lac Z transgenic model with standard methods used to assess mutation frequency and type at the native hprt locus. Age-related changes in the level and type of mutations at the hprt and lac Z locus will be determined by comparing splenic T cells from irradiated and non- irradiated mice of different ages. A single genetically defined imbred mouse strain will be used that carries a unique lac Z transgenic construct. This strain is the result of a single insertion event at the gene level, and carries a concatemer of approximately 20 lacZ constructs at a single locus. The nature of each individual construct allows for the efficient recovery of unit lac Z sequences from genomic DNA. In this study, DNA from splenic T lymphocytes will be obtained from transgenic mice at 6, 12, 18 and 24 months of age and screened for loss of functional lac Z sequences. The frequency of lac Z mutations will be measured by a loss of beta-galactosidase activity which can be assayed according to the viability of a galactose epimerase negative E. Coli C host. The study will include 2 groups: 1) animals fed standard lab chow ad lib, and irradiated daily with 150R for three days, and 2) unirradiated controls. For each of these groups, mutational frequency determined using both protocols will be carried our on the same sample for comparative purposes. Using this approach, we hope to demonstrate that the lac Z model can be used to reflect mutational phenomena at native loci over a range of ages and in response to mutagenic stresses known to mimic oxidative stresses in vivo. Validating this model would allow it to be used further as a reliable readout to investigate genetic loci responsible for age-related changes in mutation rate. Identification of such loci would eventually lead to a more definitive test of the somatic mutation theory of aging.
