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dc.contributor.authorWalker, Wendilywn Elizabethen
dc.date.accessioned2018-01-31T11:39:32Z
dc.date.available2018-01-31T11:39:32Z
dc.date.issued2005en
dc.identifier.urihttp://hdl.handle.net/1842/27597
dc.description.abstracten
dc.description.abstractCystic Fibrosis (CF) is the most common lethal inherited disease, affecting -1/2000 live births. Although the genetic lesion, a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, has been elucidated, the exact mechanism whereby this causes the debilitating disease phenotype is unclear. CF patients are prone to repeated bacterial infection of the lung; leading to fibrosis of this tissue, and eventually respiratory failure. Gene therapy has the potential to cure CF: by introducing a normal copy of the CFTR gene into epithelial cells of the lung, it may be possible to abolish the lung phenotype. While early gene therapy vectors utilised a strong viral promoter (e.g. Pcmv) to drive expression of the CFTR cDNA, it has become apparent that this approach creates problems: expression is short lived and may not be targeted to the correct cell types at appropriate levels.en
dc.description.abstractCFTR expression is tightly restricted, both temporally and spatially in affected tissues, notably the lung. Putative regulatory elements have been identified upstream of the CFTR gene, both proximaliy and distilliy. In addition, a DNase 1 hypersensitive site has been identified in intron 1 of the gene, and has been associated with an increase in CFTR expression in cells of the gut. Our approach is to include genomic components of the CFTR locus fused to cDNA in gene therapy vectors. These 'genomic context vectors' (GCVs) may be capable of recapitulating the endogenous CFTR expression pattern.en
dc.description.abstractThe 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. Smail 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. Some vectors contained an internal ribosome entry site (IRES) to allow separate translation of proteins from a single mRNA:en
dc.description.abstractThese vectors were transfected into permanent cell lines COS"/, MDCK-iowa. T84 and CaC02, in order to assess the effects of the genomic context elements upon EGFP expression. Several transfection methods were compared to optimise transfection efficiency, including the liposome DOTAP, the Saint-Mix™ synthetic amphiphile delivery system, a Polyethyieriimine (PE1) method, and the LID method. A novel transfection method, the 'SID' method (incorporating Saint-Mix ™ and the integrin targeting peptide Pb), showed early promise. Ultimately, the LID method was chosen for further studies, as this method was both, efficient and consistent.en
dc.description.abstractThe proximal CFTR 5' region in the pi kbcfproEGFP vector drove expression of the EGFP transgene at low levels in every celi 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 pacRC 1 iresEGFP and pa(PC2 ires EGFP. which differ only in the absence or presence of CFTR intron ! respectively, showed similar levels of expression in the COS7 and MDC'K-iowa cell lines. Thus, the intron I 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. An investigation of the intron 1 element in gut cell lines T84 and CaC02 was thwarted by low transfection efficiency in these cell lines, coupled with inefficient translation from the IRES.en
dc.description.abstractA comparison of pEGFP-N and pacRC2citivEGFP 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.en
dc.description.abstractPermanent cell lines form an important starting point for transfection studies, but ultimately have proved to be poor models for in vivo gene transfer. An ex vivo primary air interface sheep tracheal culture was utilised as a more realistic model. These cultures were characterised by electron microscopy, and demonstrated features of the native tracheal epithelium. A measure of transepitheliai resistance confirmed the presence of tight junctions. 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 in the ex vivo cultures, thus PAC vectors, such as GCVs, may provide a promising approach for gene therapy studies.en
dc.publisherThe University of Edinburghen
dc.relation.ispartofAnnexe Thesis Digitisation Project 2017 Block 16en
dc.relation.isreferencedbyen
dc.titleTowards gene therapy for cystic fibrosis: enhanced green fluorescent protein as a reporter of promoter activityen
dc.typeThesis or Dissertationen
dc.type.qualificationlevelen
dc.type.qualificationnamePhD Doctor of Philosophyen


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