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Title: Screening of 7.5dpc mouse cDNA libraries by molecular indexing for genes involved in anterior patterning, and in silico analysis of a novel mouse protocadherin
Author: Anderson, E.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2002
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The anteroposterior axis of the vertebrate embryo is first detected anatomically during gastrulation. However, genes are reportedly restricted to the future anterior region of the visceral endoderm (AVE) prior to overt gastrulation and have been shown to be required for anterior patterning. In this work, to identify genes specific to the endoderm, and therefore potentially involved in anterior patterning, the differential representation of 7.5dpc endoderm and mesoderm cDNA libraries was investigated using the differential display-PCR technique, molecular indexing. The putative differential representation was verified by semi-quantitative PCR and Southern blotting techniques. Of the cDNA fragments tested, the representation of 49% was confirmed. However, the approach of using molecular indexing of embryonic cDNA libraries specifically to identify genes restricted to the AVE was found to be poor. Spatial and temporal expression of isolated cDNA fragments was further investigated using in situ hybridization to whole mount mouse embryos. The in situ hybridization screen identified an unknown cDNA with a restricted expression profile, which includes a ring of mesodermal tissue in the proximal part of a gastrulating embryo and a rostral-caudal gradient of expression in the midbrain at 10.5dpc. 5' RACE PCR was used to isolate the full coding region of the restricted mRNA which consisted of 920 amino acids. Analysis of sequence similarity searches, protein motifs and protein domains was performed in silico, and the isolated mRNA was found to be a novel mouse protocadherin with similarity to human protocadherin 18. Members of the cadherin superfamily are involved in cell adhesion and are necessary during embryonic development for cell sorting, cell migration and organogenesis. The cDNA fragment isolated from the molecular indexing screen was used in cross-species in situ hybridization on sectioned human embryos. There are conserved domains of mesodermal expressions between mouse and human embryos suggesting that the mouse gene isolated in this project could be used as a model for investigating human protocadherin 18.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available