Whitaker, Duncan Andrew

2019-02-15T14:26:37Z

2019-02-15T14:26:37Z

2004

Since the discovery of the CFTR gene over a decade ago, Cystic Fibrosis (CF) has been regarded as amenable to intervention by gene therapy. The ultimate aim of gene therapy must be the correction, within cells capable of repopulating the tissue, of the genetic defect in its chromosomal context. Towards that end, a mouse model designed to evaluate the efficiency of gene correction was created, and a transgenic approach was taken to the investigation of a putative progenitor cell population in the adult murine respiratory tract. Before gene correction systems can be considered as valid therapeutic agents, their utility in the cells and tissues of living animals must be demonstrated. Thus, an in vivo system permitting the simple quantification of correction frequency in a wide range of tissues would be a valuable resource for the gene correction community. The generation and analysis of a transgenic mouse carrying an inactivated, but potentially correctable, reporter transgene is described. The full potential of a gene correction strategy to provide a single-dose, permanent solution to a genetically-diseased tissue will only be realised once the therapy is able to target resident stem cells. For CF lung disease, this will require the prior identification of stem cells in the respiratory epithelium. Previous work has indicated that potential stem cells are spatially coincident with small groups of cells expressing high levels of keratin 5 (K5) protein in the proximal murine trachea. In order to investigate lineage arising from this putative stem cell niche, transgenic mice have been generated which express an inducible form of Cre recombinase from the K5 promoter. Preliminary experiments demonstrate recombination of a conditional reporter gene after induction of Cre activity in K5-expressing tissue. Comparison of the inducible system with a constitutive K5 promoter-driven Cre line validated the choice of the former, as the clarity of data obtained from the conventional system was undermined as a result of K5 promoter activity causing reporter gene activation prior to the onset ofthe experiment. In the course of these studies it became evident that the conventional, constitutive Cre line gave rise to segregating patterns of reporter gene activation. While some mice displayed the expected K5-derived expression profile, other animals demonstrated ubiquitous expression. Universal activation of the conditional reporter was detected only in animals derived from females carrying the Cre transgene, and was found to be the result of unanticipated production of Cre protein in the maternal germline. This transgenic line is unusual and valuable in offering a choice of tissuespecific and generalised recombination of floxed alleles.

http://hdl.handle.net/1842/34403

The University of Edinburgh

Annexe Thesis Digitisation Project 2019 Block 22

Already catalogued

Transgenic approaches for the investigation of putative airway stem cells as potential targets for gene correction therapy

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy