Our laboratory has been systematically studying the functional expression patterns of the NZM2410-derived lupus susceptibility loci Sle1, Sle2, and Sle3/5 using a mixed chimera approach in which different combinations of bone marrow from congenic donors with allelically marked T and B cells are co-infused into a congenic non-autoimmune host. Mice tricongenic for these intervals, called B6.Sle1.Sle2.Sle3 have an aggressive autoimmune phenotype similar to the parental strain. Results of mixed chimera have demonstrated the potential for gene-gene interactions within a cell and between cells. These experiments also suggested that mixed chimeras may offer unique advantages in the emerging field of functional genomics and proteomics. The hypothesized advantages of the mixed chimera approach include 1) the ability to directly compare similar cell populations exposed to the same environment but differing by expression of susceptibility loci and autoimmune-related outcome (i.e. loss of tolerance); 2) the ability to compare enhanced phenotypic differences that are frequently seen in the mixed chimeras compared to unmanipulated controls; and 3) an improved ability to systematically evaluate epistatic interactions at the gene level. We therefore propose an R21 feasibility study to test these hypotheses. The following mixed bone marrow chimeras will be prepared: (1) B6.Ly5 (a) + B6 and (2) B6.Ly5 (a) + B6.Sle1 .SIe2.Sle3. ? Following demonstration of a high-titer anti-dsDNA response in the second group, mice will be euthanized and CD19+ and CD4+ cells will be isolated by magnetic beads.
Two specific aims are proposed: ? Specific Aim 1. To use a proteomics approach to identify protein expression differences; ? Specific Aim 2. To use a gene array approach to identify differences in gene expression. ? Successful completion of these experiments would identify a novel method of systematically identifying epistatic interactions in an important murine model of SLE. ? ?
