To identify new genes modulating prion resistance, we took advantage of the sheep model, where susceptibility/resistance is mainly dominated by specific allelic forms at the prion (PRNP) locus. For this purpose, two families were scanned with microsatellite markers for genes modulating scrapie resistance. The ARQ/VRQ individuals were raised in an environment where scrapie is naturally present. When the lambs reached the age of 3 and 6 months amygdales biopsies were made in order to evaluate the presence of prions, and these phenotypical data was recorded. Subsequently, the animals were observed until the appearance of the first clinical signs of the disease and, the diagnosis was systematically confirmed by an histological examination of the brain.
In parallel with the progress in the genetical refining of the QTL region, we develop a research program aiming at gene expression pattern alterations caused by scrapie in organs relevant for the disease. For this, mRNA is extracted from cerebral tissue and lymphoid tissue of sheeps of same breed, age and sex, healthy or ill. Organ-specific cDNA pools are synthesized and a differential expressed library of ~2000 clones will be constructed by Suppressive Subtractive Hybridisation (SSH). The cDNA products will be spotted on high-density nylon macroarrays, hybridise with cDNA from animals of various genotypes and disease state, before and after the apparition of symptoms. In parallel, the differentially expressed genes identified, will be mapped on the sheep genome to evaluate their position in reference to QTL regions.
This double approach (genetic mapping and functional genomics) could therefore speed up the identification of genes involved in scrapie action modulation. Furthermore, analysing the function of these genes could open the way to a better understanding of prion diseases pathogenesis. Such genes could constitute new targets for therapeutic trials and markers exploitable for diagnostic tests.
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