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Title: RAGE-based strategies for the control of gene expression
Author: Lovejoy, Elizabeth A.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1999
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The Cre/loxP site-specific recombinase system evolved within bacteriophage PI as a mechanism to maintain correct unit copy segregation of the prophage within host cells. This thesis reports the application of this system to regulate gene expression in murine cells. To regulate gene expression via RAGE (Recombination Activated Gene Expression) a novel floxed STOP cassette was designed, constructed and tested in murine embryonic fibroblasts (EF) and embryonic stem (ES) cells. When the floxed STOP cassette was used to regulate the expression of the Enhanced Green Fluorescent Protein (EGFP) marker gene a two-fold upregulation of EGFP transcription was observed after Cre mediated excisive recombination. However, no expression of the EGFP gene could be detected at the protein level and several reasons for this observation are discussed. The floxed STOP cassette was also utilised in RAGE-based strategies to achieve conditional expression of the tumour suppressor gene p53. A complex array of biological functions has been assigned to p53. For example, p53 is known to be involved in the regulation of apoptosis, multiple cell cycle checkpoints and the onset of replicative cellular senescence. The development of new approaches to achieve conditional p53 expression should be a valuable tool and permit further investigation into the pleiotropic nature of p53 function. Therefore, the floxed STOP cassette was used to regulate the expression of a p53 cDNA in p53 null primary EF cells in vitro. The upregulation of p53 expression after Cre administration was detected, but at a low frequency, by immunohistochemistry. The response of EF cells to the expression of p53 in terms of replicative cellular senescence was also characterised, including the first description of senescence-associated β-galactosidase expression in any murine cell. The floxed STOP technology was also used in an attempt to generate tools that will allow regulated expression of the endogenous murine p53 gene.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available