This project has two principal goals: (1) to identify regions of the mouse genome that encode genes that contribute significantly to longevity; and (2) to use selective breeding strategies to produce strains of mice that age at different rates. The experiments will utilize genetically heterogeneous mice bred by a four-way Cross between (C57BL/6 x SJL)F1 females and (AKR x DBA/2)F1 males (""""""""HET"""""""" mice). The analytical genetic study will begin by typing 200 HET mice at each of 190 informative loci, using PCR-based amplification of primer pairs that discriminate among 2 or more of the grandparental inbred strains. A second cohort of 200 HET mice will then be genotyped using the original battery of primer pairs plus a second set of 95 probes selected to provide higher resolution in regions suggested to be of particular interest during the initial screen. Mice in the second cohort will also be tested at 6 and 18 months of age for indices of age-related immune decline: the proportion of memory T cells within the CD4 helper and CD8 killer T cell subsets. Each of the HET mice will be sacrificed when moribund to obtain longevity and pathology data. An interval-based mapping strategy will then identify quantitative trait loci (QTL) that influence longevity. Parallel analyses will establish whether these, or other QTL, also influence the incident rates of major disease processes, or influence the rate of physiological aging as measured by shifts in T lymphocyte subset composition. The selective breeding study will generate lines of mice, starting from HET stock, based on differences in (a) early life hemagglutinin response; or (b) distribution of reproductive effort. These selective criteria have been chosen because each can be assayed within the first 8 months of life, and because theoretical and experimental considerations suggest that each trait may be influenced by genes that pleiotropically affect aging rate and hence longevity. The selection protocols incorporate independent replication (bidirectional for the hemagglutinin protocol) and parallel development of unselected control stocks for genetic and phenotypic comparison to the selected lines. When breeding has produced lines of mice that differ in the selected criteria, groups of animals from each selected line will be set aside for determination of longevity, pathologic study, and genotyping using primer sets developed by analysis of the HBT stock. RNA from old and young HET mice, and later from selectively bred animals, will be provided to Dr. Judith Campisi for a collaborative study of age- related alterations in gene expression in monotypic cell preparations. Long-term goals include (a) selection of mice based on genotype at QTL identified through analysis of HET mice; (b)positional cloning of genes within regions identified by QTL analysis of HET and selected mice; and (c) detailed physiological analysis of proposed anti-aging defenses in mice of short- and long-lived genotypes.
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