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Title: Studies on conditional gene expression
Author: Lyons, Scott K.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1999
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Using a double replacement targeting approach, I attempted to replace the p53 promoter region with inducible promoter sequence. Targeting constructs were designed to produce a mutant p53 locus, which would then subsequently allow the rapid and simple introduction of a variety of transcriptional regulatory sequences. The first stage vector was introduced into ES cells, and of 508 HAT resistant clones screened, 1 clone possessed a recombinant p53 locus. Time constraints did not permit full characterisation of this clone, either in vitro or in vivo. In a parallel approach, I used one of the candidate promoter systems to generate an interferon α/β inducible p53 transgene. Sarcoma cells, derived from an engineered homozygous p53 deficient mouse, were established as lines in vitro and stably transfected with this construct. Analysis of resultant inducible clones demonstrated little significant transgene mediated effect upon tumour cell cycle. However, a clear transgene dependent apoptotic phenotype was observed following UV irradiation. To test the possible extent of therapeutic benefit resulting from the introduction of p53 gene expression into p53 null tumour cells in vivo, 5 x 106 inducible sarcoma cells were injected sub-cutaneously into the flanks of SCID animals. Tumours grew at the site of injection within 3 weeks, at which time the animals were injected intra-peritoneally with synthetic double stranded RNA (pI:pC) to induce transgene expression. No p53 protein could be detected in these tumour samples by either western or immuno-histochemical analyses. Subsequent re-establishment and analysis of cell lines derived from these tumours indicate that, during the clonal expansion of the original inoculate in the SCID animal, selection had occurred for a final presenting tumour cell with lost or silenced p53 transgene expression. Following pI:pC injection of SCID mice, an acute apoptotic phenotype was observed in the crypt compartments of the small intestine which resulted in the death of approximately 20 % of all injected animals.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available