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Title: Intestinal epithelial stem cells as targets for CF gene therapy
Author: Slorach, Euan M.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1998
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As a first step towards mutation correction by homologous recombination I have constructed a targeting vector which contains wild type Cftr genomic DNA covering the region mutated in the majority of CF mouse models and shown that it is capable of targeting the Cftr locus in ES cells. In order to test this strategy in a more relevant system, I have generated several cell lines from the small intestine of both wild type and CF mice carrying the Cftrm1HGU mutation. These lines have been characterised and used in a pilot targeting experiment. The absence of intestinal stem cell markers necessitates the need for a functional assay in order to identify stem cells and ensure that any manipulation does not alter their stem cell properties. I have developed such an assay using aggregates of mouse intestinal cells which when grafted into a suitable host, develop into cysts lined with an intestinal mucosa containing all four epithelial cell lineages and full crypt-villus architecture suggesting the presence of intestinal stem cells in the original population. Detailed characterisation of graft development revealed a process highly similar to regeneration of intestinal epithelium following tissue damage and suggests the continued presence of a stem cell population. Intestinal epithelial stem cells will need to be maintained in tissue culture if an ex vivo approach to gene therapy is to be successful. To this end, I have established primary cultures of mouse intestinal epithelium and shown that two different proliferative epithelial colonies can be identified. Characterisation of these colonies suggests that they are derived from two different proliferative epithelial populations with properties similar to those of transit amplifying cells and stem cells of the mouse small intestine.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available