Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.604947
Title: Stem cell proliferation and lineage analysis in the small intestinal epithelium : a transgenic approach
Author: Ireland, H.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2005
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Abstract:
The epithelial lining of the small intestine is continuously renewed by a small population of pluripotent cells that are poorly characterised. This thesis describes the development and application of a novel clonal marking approach to investigate their properties. The experimental approach employs a somatically activatable reporter based on the regulated expression (from an inducible cytochrome P450 promoter) of Cre recombinase. This approach is employed to address two questions of fundamental biological importance. How often do intestinal stem cells divide? Do Paneth cells, one of the four main intestinal types, arise from multipotent or unipotent precursors? To investigate the frequency of stem cell division, transgenic animals were generated with a cre fusion gene containing an unstable dinucleotide repeat tract that places the cre out-of -frame with respect to its coding sequence. Expression of cre occurring as a result of error prone DNA replication restores the reading frame and switches on a reporter, thus marking the affected cell. Because such frame shift mutations arise as a consequence of DNA replications they will occur at a frequency proportional to the total number of cell divisions. Hence, over time, mutant reporter expressing clones will accumulate where the target population are both proliferative and retained for long periods: as is the case for stem cells. Sampling aged cohorts of transgenic animals on an appropriate mismatch repair deficient background revealed a linear accumulation of mutant clones at a rate determined to be 6.5 - 8.3 x 10-4 clones/transgene/month and that allowed the calculation of the mitotic rate of intestinal stem cells. For lineage analysis the inducible cre was fused to a mutated oestrogen receptor allowing precise control of recombination frequency. Marked clones comprising a single cell type, the Paneth cell, were identified and analysed in cohorts of induced mice. These investigations led to a further understanding of Paneth cell lineage and clearly demonstrated that their lifespan under normal physiological conditions is twice as long as previously reported.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.604947  DOI: Not available
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