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Title: The molecular characterisation of a gene trap integration into the Aminopeptidase O (ApO) gene
Author: Axton, Richard Alan
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2006
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The protease aminopeptidase O (ApO) was isolated during a gene trap screen designed to identify novel developmentally regulated genes involved in cardiovascular development. The aims of this thesis were to characterise the gene trap integration, determine the full-length open reading frame (ORF) and to investigate any functional domains that the gene contained. Additionally, germline transmission of the gene trap demonstrated expression in the vasculature of mice. Work was undertaken to investigate if there was a phenotype in the homozygous gene trap mice with particular emphasis on whether this gene was involved in the process of new blood vessel formation (angiogenesis). Initially, 5' RACE (Rapid Amplification of cDNA Ends) generated a 206 bp sequence that aligned with expressed sequence tag data to build a contig with a partial ORF of 177 amino acids. Additional rounds of 5'RACE generated a full length open reading frame of 823 amino acids. The nucleotide and protein sequence are highly conserved between mouse, rat and human. The gene encodes a catalytic domain characteristic of the Ml family of aminopeptidases and contains a C-terminal armadillo repeat sequence and a putative nuclear localisation signal (NLS). The NLS was experimentally confirmed by transfection of eGFP-ApO constructs demonstrating that the signal localised the fusion protein to the nucleoli. The gene trap ES cells were injected into blastocysts and transmitted through the germline. Generation of homozygous animals resulted in normal, healthy, viable mice with no apparent phenotype. Expression analysis by lacZ staining and RNA in situ indicated that ApO expression was restricted to the cardiovasculature of embryos and adult tissues. The possibility that the ApO may be involved in the process of angiogenesis was investigated using the aortic ring assay. Northern blot analysis has shown that there are tissue specific isoforms and RT-PCR analysis has revealed that the catalytic domain and nuclear localisation signal can be removed by alternative splicing. An antibody was raised against purified full length murine APO protein and the immunopurified antibody tested by Western blot, immunoprecipitation and immunohistochemistry.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available