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Title: Examination of the role of the Wilms' tumour suppressor gene (Wt1) in mouse development and neoplasia using a homozygous Denys-Drash syndrome mutation
Author: Wagner, Kate J.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2001
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Denys-Drash syndrome, which is characterised by genitourinary abnormalities, a type of progressive nephropathy known as mesangial sclerosis and the development of Wilms' tumours, results from mutations in the zinc finger-coding region of one copy of the WT1 gene. Murine ES cells from the CGR8 line with DDS-type mutations in both Wt1 alleles (compound heterozygotes) were produced by gene targeting and their properties compared with wild-type and DDS heterozygous cells. To distinguish between effects of the Wt1 mutations and of alterations elsewhere in the genome, cells of the E14 ES cell line and their DDS heterozygous and homozygous derivatives were also studied. While attempting to characterise the mutant cells in terms of RNA and protein expression, it was discovered that undifferentiated ES cells do not express Wtl. To enable the detection of mutant and wild-type Wtl RNA transcripts and proteins, a previously published method of ES cell differentiation using all trans-retinoic acid was adapted to optimise Wtl expression. Examination of Wtl expression levels in differentiated cells of the different DDS genotypes revealed a reduction in the level of mutant protein in the DDS heterozygotes and compound heterozygotes compared with that predicted from the relative levels of RNA transcripts. Embryoid bodies were produced using cells of the above genotypes. Some of these were grown in suspension, others were allowed to attach to the surface of a flask and form outgrowths. A comparison of these embryoid bodies revealed no obvious differences between the differentiation capabilities of wild-type and DDS mutant ES cells. As abnormalities of Wtl have been found in leukaemia, including expression of DDS-type mutations, more embryoid bodies were produced, and their potential to differentiate along haematopoietic lineages was assessed using the CFU-A assay. This revealed a delay in the production of haematopoietic cells in Wtl mutant embryoid bodies relative to those produced from wild-type cells, which was consistent between the two cell lines examined. Further evidence for this delay was obtained by the examination of the expression patterns of several genes known to be involved in haematopoiesis, using RT-PCR.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available