Immune-mediated disorders are an important cause of renal disease in children and adults. Autoimmune processes, involving the production of autoantibodies with high affinity and specificity for the inciting autoantigens, are often implicated in the pathogenesis of such disorders. The process of somatic hypermutation is intimately involved in the generation of such affinity antibodies, both protective and autoreactive, yet, little is known of the nature or mechanisms of this process. The present proposal, under the sponsorship of Dr. Richard Barth, Director of the Transgenic Facility, University of Rochester, will provide the candidate with training in the use of transgenic animals to study the basic characteristic of somatic hypermutation. A transgene will be constructed and contain a non-expressed rearranged human V 6 immunoglobulin heavy chain gene which will be incorporated by homologous recombination into the murine heavy chain locus of mouse embryonic stem cells. Transfected embryonic stems cells will be injected into blastocysts derived from Recombinase Activating Gene-2 deficient mice. The B cells of these chimeric mice will be derived from the tranfected embryonic stem cells. These mice will be heterozygous for the transgene but should rearrange endogenous V genes at the other heavy chain locus. Both alleles will be subject to somatic hypermutation upon subsequent immunization. Hybridomas will be made from spleen cells, cloned, and supernatants screened for reactivity with the immunogen. cDNA will be produced form positive clones, amplied with transgene specific primers and the nucleotide sequence determined. The characteristics of the somatic mutations introduced into the transgene, which will be uninfluenced by antigen selection, will be determined. Specific aspects of the mutations of germline nucleotides leading to transitions vs. transversion, bias in the strand of DNA mutated, and bias in the distribution of mutations in the rearrangement.
| Varade, W S; Carnahan, J A; Kingsley, P D et al. (1998) Inherent properties of somatic hypermutation as revealed by human non-productive VH6 immunoglobulin rearrangements. Immunology 93:171-6 |
| Insel, R A; Varade, W S (1998) Characteristics of somatic hypermutation of human immunoglobulin genes. Curr Top Microbiol Immunol 229:33-44 |
