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RAGE-Based strategies for the control of gene expression

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LovejoyEA_1999redux.pdf (52.37Mb)
Date
1999
Author
Lovejoy, Elizabeth A.
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Abstract
 
 
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 achieve the regulation of gene expression a novel floxed STOP cassette was designed, constructed and tested in murine embryonic fibroblasts (EF) and embryonic stem (ES) cells. It was shown that the floxed STOP cassette could be used successfully to regulate the transcription of the Enhanced Green Fluorescent Protein (EGFP) marker gene in ES cells. 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 strategies to achieve conditional expression of the tumour suppressor gene p53. A complex array of biological functions have 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 novel 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 (3-galactosidase expression in any murine cell. The floxed STOP technology was also used in an attempt to regulate the expression of the endogenous murine p53 gene in vivo. The successful integration of the floxed STOP cassette, via an inducible gene targeting strategy, into the murine p53 locus of ES cells is reported.
 
Two methods were explored to achieve regulated expression of Cre recombinase in vitro, the novel technology of recombinant adenoviridiae and the identification of embryonal stem cell differentiation state-specific promoter regulatory sequences.
 
In conclusion, this thesis has explored the feasibility of using Cre/loxP-based technologies to regulate gene expression. Several test plasmids were constructed and characterised and all demonstrated that the application of floxed STOP-based approaches is not as straightforward as suggested by the relatively small body of literature in this field.
 
URI
http://hdl.handle.net/1842/26699
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