Controlled gene expression using acute phase response elements

Harraghy, Niamh (2001) Controlled gene expression using acute phase response elements. PhD thesis, University of Glasgow.

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Abstract

Previous work in our laboratory involved the creation of an inducible gene expression system based on the promoter of the major acute phase protein in humans, C-reactive protein (CRP). The aims of this project were to further characterise the CRP based expression vector and to modify it for use in pigs. The green fluorescent protein (GFP) was also evaluated as a reporter of transient inducible gene expression. The 30kb fragment containing the human CRP gene was sequenced and analysed for the presence of elements that may be responsible for the low basal levels of expression of the gene and for sequences that are responsible for the sexually dimorphic pattern of expression of the human CRP gene in transgenic mice. It was planned to use the information from these analyses to modify the expression vector for use in pigs. However, because it is not currently known how the CRP- based acute phase expression vector would behave in other species, the promoter of the major acute phase protein in pigs, ITIH4, was isolated. Due to the absence of homology between the pig ITIH4 promoter and the human CRP promoter, the pig ITIH4 promoter was further characterised. The investigations focused on the inducibility of the promoter in response to cytokine stimulation and the effect modification of the promoter would have on promoter activity and inducibility. It was found that the promoter was induced by IL-6 but not IL-1 (except at low concentrations). A combination of IL-1 and IL-6 was shown to result in a decrease in the inducibility of the construct by IL-6. Mutation of the promoter in order to decrease the basal level of expression and enhance inducibility was unsuccessful. In order to develop a system that will facilitate studies of inducible gene expression in vitro a destabilised variant of GFP was evaluated as a reporter gene. Although small increases in fluorescence intensity could be detected following stimulation, the analyses suggest that GFP is not as sensitive as other reporter genes for studying inducible gene expression.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Advisers: Tim Mitchell; Gary Evans; Richard Wales
Keywords: Genetics
Date of Award: 2001
Depositing User: Enlighten Team
Unique ID: glathesis:2001-72386
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 24 May 2019 15:12
Last Modified: 24 May 2019 15:12
URI: http://theses.gla.ac.uk/id/eprint/72386

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