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Title: The characterisation and expression of ovine MHC class-II genes
Author: Ballingall, Keith Thomson
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1991
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The Major Histocompatibility Complex (MHC) is a multigene complex encoding polymorphic cell surface glycoproteins involved in the initiation and regulation of specific cellular immune responses to foreign antigen. Within the class-II region of the human and murine gene complexes are a large number of alpha and beta genes which encode dimeric cell surface glycoproteins (MHC class-II molecules) involved in the presentation of exogenous foreign antigen to cells of the immune system. In an attempt to characterise these genes and their products in the sheep, two genomic cosmid DNA libraries had previously been constructed. These libraries were screened with human and murine class-II alpha and beta genes as probes. Seven distinct alpha and twenty four beta genes had been identified. The aim of this project was to identify combinations of these alpha and beta genes that co-express following DNA-mediated gene transfer into mouse L-cells. All combinations of alpha and beta genes were transfected along with the thymidine kinase gene as a selection marker into the Tk and class-II deficient mouse L-cell LTk-. Following HAT selection surviving cells were analysed for cell surface ovine class-II molecules by an indirect fluorescence antibody assay and FACScan analysis. Following transfection of four combinations of two alpha and two beta genes ovine class-II molecules were detected at the L-cell surface. These all represented an ovine equivalent of an HLA-DR molecule (OLA-DR) as determined by Southern hybridisation and nucleotide sequence analysis. L-cell lines expressing high levels of OLA-DR were developed by FACS sorting and expansion in culture. These lines were employed to type two panels of monoclonal antibodies specific for monomorphic determinants of ovine MHC class-II molecules for OLA-DR specificity. Cell surface OLA-DR specificity was attributed to eight out of twenty four monoclonal antibodies by an indirect fluorescence antibody assay. These results were confirmed and in addition alpha or beta chain specificity determined by use of an immunoblotting technique on lysate prepared from each transfected L-cell line.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available