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.
