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Title: Analysis of transient gene expression in ovine cells : a role for the PrP gene 3'UTR
Author: Marshall, Elaine
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2000
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In a previous thesis, in vitro expression in mouse N2a cells using reporter gene constructs with the chloramphenicol acetyl transferase (CAT) gene linked to PrP sequences had suggested that there might be an inhibitor of translation in the PrP gene 3'UTR. Transient transfection methods were developed to generate levels for in vitro expression of the CAT/PrP-3'UTR constructs in immortalised Cheviot fetal brain-derived cell lines from both scrapie resistant and susceptible genotypes and primary cell lines derived from cerebellum and liver tissues of the Icelandic sheep breed, Ovis brachyura borealis pall. Expression of a series of CAT/PrP-3'UTR vectors in cell lines derived from different PrP genotypes found that sequence between nucleotides 2000-2700 on the PrP 3'UTR may show a tendency to reduce protein expression in the cells derived from brain tissue of scrapie-resistant genotype genotype and in a tissue specific manner. Additional vectors were produced to express ovine PrP mRNAs, similar to the endogenous 4.6kb and 2.1 mRNAs transcripts, but altered to display a monoclonal epitope site (3F4) enable transiently expressed PrP protein within ovine cell cultures to be detected. Expression of the PrP constructs in mouse neuroblastoma (N2a) cells has shown that both constructs were viable. However, the immunodetection methods employed in this thesis could not distinguish between transiently expressed 3F4-labelled ovine PrP and endogenous PrP within sheep cell lines. Results presented here confirm that the 3' UTR found in the 2.1 kb mRNA is capable of supporting gene expression. A role for a specific sequence in the ovine PrP gene 3'UTR in controlling protein expression in brain-derived cell lines has been proposed. Also, the function of the regulatory sequence may be dependent on tissue origin. PrPc is vital for the replication of the TSE agent. Controlling the amount of available PrPc in vivo may influence susceptibility and development of TSE disease.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available