Optimisation of the ARF5 transcription factor for use in a novel gene regulation system

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dc.contributor.advisor Young, D en
dc.contributor.author Aspell, Tiger en
dc.date.accessioned 2018-07-03T00:19:03Z en
dc.date.issued 2018 en
dc.identifier.uri http://hdl.handle.net/2292/37373 en
dc.description Full text is available to authenticated members of The University of Auckland only. en
dc.description.abstract In recent years, a lot of research has focused on gene therapy as an alternative treatment for neurodegenerative diseases. A major problem with most existing gene therapies, however, is that the therapeutic gene (transgene) packaged inside viral vectors is under the control of a constitutively active promoter. This means there is high, unregulated activation of the transgene. In terms of using such systems in the clinical setting, there are high risks of adverse side effects arising from the over-production of therapeutic protein. The aim of this thesis was to optimise the ARF5 transcription factor in the novel regulatory system developed in the Young Laboratory, responsible for increasing gene activity in times of cell stress and returning this activity to baseline levels under normal physiological conditions. It was hypothesised that the addition of different activation domains (p65 and HSF1) to plasmids containing ARF5 and a GFP reporter gene would increase GFP expression above that seen from the original backbone plasmid. However, immunocytochemical and Western Blot analyses rejected this hypothesis, and GFP expression was not significantly increased. Furthermore, it was found that the addition of a nuclear localisation signal (NLS) did not promote nuclear entry of ARF5. Upon the excision of the WPRE and micro luciferase (miLucx3) molecules from the plasmid containing p65 and HSF1, however, GFP expression was significantly increased, almost to the levels observed in cells transfected with the empty control plasmid. Collectively, these results suggest that the addition of activation domains and the removal of a WPRE-miLucx3 sequence significantly increase gene activity. Finally, the VP64-p65-HSF1 plasmid was determined to be the one which expressed the most GFP and could, potentially, be the plasmid which activates a therapeutic gene in the Young Laboratory’s regulatory system. en
dc.publisher ResearchSpace@Auckland en
dc.relation.ispartof Masters Thesis - University of Auckland en
dc.relation.isreferencedby UoA99265072012102091 en
dc.rights Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. Previously published items are made available in accordance with the copyright policy of the publisher. en
dc.rights Restricted Item. Available to authenticated members of The University of Auckland. en
dc.rights.uri https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm en
dc.rights.uri http://creativecommons.org/licenses/by-nc-sa/3.0/nz/ en
dc.title Optimisation of the ARF5 transcription factor for use in a novel gene regulation system en
dc.type Thesis en
thesis.degree.discipline Medical and Health Sciences en
thesis.degree.grantor The University of Auckland en
thesis.degree.level Masters en
dc.rights.holder Copyright: The author en
pubs.elements-id 746890 en
pubs.org-id Medical and Health Sciences en
pubs.org-id Medical Sciences en
pubs.org-id Physiology Division en
pubs.record-created-at-source-date 2018-07-03 en

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