MOLECULAR AND BIOCHEMICAL ANALYSIS OF THE CANINE MHC

Thomas Jefferson University, Philadelphia PA

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Abstract

DESCRIPTION (Adapted from the applicant's abstract): Canine models of human diseases have been used to study autoimmune thyroiditis, gluten-sensitive enteropathy, narcolepsy, complement deficiencies, and solid organ and hematopoietic progenitor cell transplants. Of paramount importance to comprehending immune-related diseases and transplantation immunology is an understanding of the MHC. In spite of the dog's usefulness in research, molecular and biochemical analyses of the canine MHC (termed DLA) have lagged behind those of the human MHC (HLA) and the murine MHC (H2) regions, as well as the MHC regions of various agricultural animals. The objective of this application is to develop a comprehensive understanding of the structure, polymorphism, and function of the canine class I and class II MHC genes. The specific goals are: 1) develop a DNA-based histocompatibility typing system for polymorphic class I and class II loci applicable to all dogs; 2) determine the structure and organization of the canine MHC by molecular mapping; and 3) identify tissues expressing class I proteins using newly generated monoclonal antibodies (MAbs). Histocompatibility typing for class I and class II genes will be performed using locus specific amplification and separation of alleles by single-strand conformational polymorphism (SSCP) gels. Pulsed-field gel electrophoresis (PFGE) with locus-specific probes will be used to determine the molecular organization of the DLA class I and class 11 gene families. A cosmid library will be constructed from a canine-hamster hybrid cell line containing a few dog chromosomes, one of which contains the DLA region. This library will be useful for mapping as well as identifying new genes within the DLA region. Class I expression will be analyzed at the protein level. Cell lines expressing canine class I genes will be produced, and reactive MAbs generated to be used in fluorescent activated cell sorting (FACS), immunoprecipitation, and immunohistochemistry studies. Class II expression will be initially studied using existing cross-reactive MAbs.