Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.582581
Title: Towards the development of a cell line stably propagating human prions
Author: Sutton, Liza Marie
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 2007
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Abstract:
Prion diseases are neurodegenerative disorders affecting both animals and humans. They may be sporadic, infectious or genetic in origin. Variant CJD (vCJD) is a novel form of human prion disease, experimentally proven to be acquired from the consumption of bovine prion-infected material. The causative transmissible agent of prion disease is thought to consist predominantly of an abnormal isoform of the host-encoded prion protein (PrP(), designated PrPs. To date, most prion research has been undertaken in animal models since transmitting the disease in cell culture has proved difficult. Nevertheless, a number of prion-propagating mammalian cell lines do exist but despite world-wide effort, no cell lines have shown susceptibility to human prion infection. The aim of this thesis was to develop a cell line that stably propagated vCJD prions. First, a high-throughput screening methodology for detection of human prions in cells was established. This involved subcloning of vCJD prion-infected cell lines, in order to isolate a rare susceptible clone, followed by detection of PrP using the scrapie cell assay or cell blot assay. A panel of cell lines was selected to best mimic in vivo disease and screened for susceptibility to vCJD prion infection, but mammalian neuroblastoma, neuroglial, and epithelial cell lines were unable to stably propagate human prions. Transient vCJD prion propagation (between one and six passages) was observed in two human neuroblastoma lines (SKN-SH and SHSY5Y) and a rabbit epithelial kidney line (RK13) overexpressing human PrPc. Attempts to prolong prion propagation in a human neuroblastoma cell line with retroviral infection, to mimic the intercellular spread of prions via exosomes, were unsuccessful. The second approach involved the humanisation of a mouse prion-propagating cell line, N2a. To eliminate dominant-negative inhibition of human prion propagation by murine PrPc, the endogenous PrPc was knocked-out using RNAi technology. Greater than 90% murine PrPc knockout was achieved in several N2a-derived lines and exogenous expression of mouse PrPc demonstrated that these cells were still susceptible to mouse prion infection. Human PrPc was subsequently expressed in these murine PrPc knockout lines to various levels. Following infection with vCJD prions, no long-term vCJD prion propagation was observed in these lines. A single murine PrPc knockout line overexpressing human PrP1 (two-fold) transiently propagated vCJD prions but this was not stable. The third approach used a v-myc immortalised human foetal neural stem cell (NSC) line derived from human ventral mesencephalon (ReNcell 197VM). These undifferentiated 197 cells were resistant to vCJD prion infection. Upon differentiation, the 197 cultures acquired a neuronal phenotype. Infection with a purified vCJD prion-infected brain homogenate yielded propagation of vCJD prions in these differentiated human neurons for >40 days in vitro. This work has improved understanding of the complex factors underlying cellular susceptibility to prion infection. The primary human neuronal model will be invaluable for the study of the pathophysiology of human prion disease.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.582581  DOI: Not available
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