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  Student Research Symposium Oct 2006  
  Development of a Sheep Cell Culture System for the Study of Scrapie

James Stanton and Timothy Baszler
Department of Veterinary Microbiology and Pathology

A fundamental mechanism in the pathogenesis of animal and human prion diseases is the conformational conversion of a normal host-encoded prion protein from a non-infectious form not associated with disease (PrPC) to an infectious, disease-associated form (PrPSc) that accumulates in cells of affected hosts, especially in the nervous system.  The precise mechanism of conversion is not known and it is unknown if accessory molecules, in addition to the prion protein, aid the conversion process.  Scrapie is a naturally-occurring prion disease of sheep in which the disease-associated prion protein (PrPSc) accumulates in cells of nervous, reproductive, and lymphoid systems.  The goal of this project is to use comparative transcriptomics on scrapie permissive sheep cell lines to identify possible accessory proteins necessary for PrPSc accumulation.  The hypothesis to be tested is that PrPSc accumulation requires an accessory protein(s).  The first step to test this is hypothesis is the development of a sheep derived cell culture system that is permissive to the conversion and accumulation of PrPSc.  Use of a permissive cell culture system will allow experimental conditions that reduce the number of variables that might lead to non-scrapie-associated differential gene expression.  Microglial cells (“resident” brain macrophages) have been chosen because they demonstrate in vivo PrPSc accumulation and these cells may also play a role in the neuropathology of prion diseases.  Cells derived from sheep fetal brains have been isolated and grown in culture.  Brain cells from sheep with have been inoculated with PrPSc positive material and have demonstrated accumulation of PrPSc over several passages.  Some of the PrPSc accumulating cells were characterized as microglial cells by morphology and lectin cytochemistry (positive for Ricinus communis-I).  Current efforts are focused on immortalizing the PrPSc accumulating sheep cells while maintaining prion accumulation over time.  Ideally, clones of these cell lines would then be used for comparative transcriptomics.

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