Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.662323
Title: The development of genetic models to understand the role of the Wilms' tumour suppressor gene, WT1
Author: Spraggon, L.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2003
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Abstract:
This thesis describes the use of gene targeting in embryonic stem cells, to develop two different genetic systems to facilitate the investigation of WT1 function. Firstly, using the Cre/LoxP system a conditional null allele for WT1 was generated by targeting the WT1 locus in embryonic stem cells. LoxP sites were positioned either side of exon 1 of WT1, and correctly targeted embryonic stem cells were used to generate mice. Together with tissue-specific transgenes directing expression inducible and non-inducible forms of Cre recombinase in vivo, this conditional WT1 mouse model will provide a powerful tool to further investigate the role of WT1 in development and disease. Secondly, WT1 null embryonic stem cells were generated through sequential gene targeting of both alleles of WT1. These WT1 null embryonic stem cells were used to identify endogenous genes regulated, directly or indirectly, by WT1 and also to identify interacting protein partners. To identify candidate WT1 target genes, WT1 null embryonic stem cells were studied in an in vitro differentiation system. Analysis of gene expression profiles using Affymetrix Genechip arrays, identified a set of differentially expressed transcripts, which may represent WT1 regulated genes. Interestingly, these included Wnt-4 and BF-2, two genes crucial for the development of the kidney. The biological activity of WT1 is modulated by its interaction with several known protein partners. To identify other protein partners, in vitro differentiation of embryonic stem cells, followed by immunoprecipitation of endogenous WT1 associated complexes coupled to MALDI-TOF analysis, was used to identify interacting protein partners. From this study, an interaction ofWT1 with heterogeneous nuclear ribonucleoprotein U (hnRNP U) was identified, a protein involved in transcriptional repression, the packaging and processing of RNA, and chromatin remodelling. Using a selection of lineage specific markers on differentiating embryonic stem cells, it was observed that WT1 is expressed in neuronal progenitors in vitro. Extending the analysis in vivo, WT1 was expressed in the ventral half of the neural tube in a domain that corresponds to location of neuronal progenitors. Specifically WT1 expression was detected in a progenitor domain that gives rise to motor neurons. WT1 expression was also present in motor neurons as shown by co-staining with Islet-1. The expression of WT1 in the progenitors of the neural tube opens the possibility that WT1 may have role in patterning of the neural tube. In summary, the development of these two systems will provide important resources to further understand the function of WT1. The WT1 conditional null mouse will permit the role WT1 in vivo to be examined, whilst the WT1 null ES cells provide an in vitro experimental system in which to screen for candidate WT1 target genes and interacting protein partners.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.662323  DOI: Not available
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