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Title: Towards gene therapy for cystic fibrosis : enhanced green fluorescent protein as a reporter of promoter activity
Author: Walker, Wendilywn E.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2005
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The aim of this project was to investigate the use of the Enhanced Green Fluorescent Protein (EGFP) as a reporter of CFTR promoter activity. six vectors were created coupling portions of the CFTR locus to EGFP in GCVs. Small plasmids were made by conventional cloning procedures, while large PAC vectors were made by a double recombination method employing both homologous and Cre recombinase/loxP recombination. These vectors were transfected into permanent cell lines COS7, MDCK-iowa, T84 and CaCO2, in order to assess the effects of the genomic context elements upon EGFP. The proximal CFTR 5’ region in the p1kbcfproEGFP vector drove expression of the EGFP transgene at low levels in every cell line analysed. This is in agreement with previous reports that show basal levels of CFTR expression driven by this proximal ‘housekeeping’ region. The additional upstream region in the PAC65bcfproEGFP vector did not appear to modulate expression in any of the cell lines analysed. A comparison of the twin vectors PACRC1iresEGFP and PACRC2iresEGFP, which differ only in the absence or presence of CFTR intron 1 respectively, showed similar levels of expression in the COS7 and MDCK-iowa cell lines. Thus, the intron 1 element does not seem to alter expression in these non-gut cell lines; this is consistent with reports that show regulation of CFTR expression in response to the intron element to be specific to cells of the gut epithelium. A comparison of pEGFP-N and PACRC2cmvEGFP revealed that large PAC vectors show an intrinsic reduction in expression in comparison to their small plasmid counterparts. Further experiments showed that this was not an effect of vector copy number, and that the effect could not act in trans upon a co-transfected molecule. These studies also revealed an unexpected interaction: diluting a reporter plasmid with an anonymous plasmid may actually increase its transfection efficiency. An ex vivo primary air interface sheep tracheal culture was utilised as a more realistic model. Cultures were transfected with several of the genomic context vectors. While PAC vectors had shown a dramatic reduction in expression relative to their small plasmid counterparts in the in vitro studies, only a small reduction was seen to the ex vivo cultures, thus PAC vectors, such as GCVs, may provide a promising approach for gene therapy studies.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available