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Student Research
Symposium Oct 2006 |
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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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