INDICATOR TRANSGENIC MICE FOR LYMPHOCYTE AGING ANALYSIS

Scripps Research Institute, La Jolla CA

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Abstract

DESCRIPTION: This project will examine whether several basic parameters of B lymphocyte function and development change with age. The project plans to use both normal and transgenic mice for these analyses. Three major questions are: 1) whether the extent and nature of Ig gene recombination events that occur during various stages of B lymphogenesis change with age; 2) whether the rate and selective success of antigen-driven somatic hypermutational processes are altered with age; and 3) whether division rates within defined mature B cell subsets change with age. The first of these will be approached through the analyses of J-kappa segment and/or C-lambda representation among B lymphocyte subsets from normal (BALB/c) mice, as well as from 3-83 transgenic and/or knock in mice. These analyses will employ PCR and related analyses with appropriate primers on either whole splenic B cells or isolated B cells. The relative representation of J-kappa segments and lambda will be compared and the degree of skewing towards C proximal J kappa segments or lambda usage will be interpreted as evidence of receptor editing events during development. The transgenic studies are designed to distinguish antigen driven "peripheral" editing versus events that occur developmentally, by introducing the 3-83 ligand. The second aim focuses on whether the rate and/or appropriate selection of beneficial somatic mutations that occur during late primary responses change with age, and whether this can be offset through hyper mutation (possibly distinguishing whether it is "rate" versus the "selection" system that has changed). These studies will use a novel set of transgenic mice that should afford "counting" the representation of somatic mutants in various B cell subsets before, during, and after antigen-driven stimulation. The studies outlined in the third aim rely on the development of a novel transgenic mouse that will permit detection of episomal elements similar to those generated during VDJ recombination. In these studies, it is envisioned that only cells which do not divide subsequent to initial maturation will retain these episomal elements in reasonable quantity, and thus the relative proportion of cells which have not divided since initial maturation can be assessed by engineering a GFP expression system that relies on the episomal excision product's persistence.