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Title: Development of a subtractive hybridization technique and the cloning of the mouse epsilon isoform 14-3-3 protein, a gene involved in fetal kidney development
Author: McConnell, Jane E.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1994
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The aim of this project was to devise a simple and efficient method to identify cDNAs expressed specifically in the mouse fetal kidney which might be important in kidney development. A 14.5 day post coitum fetal kidney cDNA library was made which has a depth of 1.2 x 106 original clones and an average insert size of 1.5 kilobases. Using a novel subtractive hybridisation technique which identifies genes expressed in the fetal kidney but not in the adult liver, a subtracted 14.5 day post coitum fetal kidney cDNA pool was obtained. By screening the library with the subtracted fetal kidney cDNA, several clones were identified. Six, when analysed by sequencing and northern blot analysis, were shown to contain B1 embryonic repeats and were not analysed further. Three, when looked at by northern blot analysis and sequence homology, proved to be the most interesting. Further characterisation identified the clones as three independent isolates of a cDNA encoding the ε member of the 14-3-3 gene family, implicated in the regulation of protein kinase C activity and of exocytosis. Northern blot analysis of RNA identified a 2.0 kilobase transcript present in many tissues including the fetal kidney, but not in adult liver. More detailed in situ hybridisation analysis showed that, although the gene is expressed in most 12.5 day post coitum embryonic mouse tissues, the level of expression varies. In general, it is low in epithelial tissues and high in mesenchymal cells. As the mesenchyme in bone, kidney, gut and lung differentiates, the level of expression drops. This observation argues that the 14-3-3 ε isoform protein plays a role in mesenchyme differentiation including acting as the kidney mesenchyme epithelialises.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available