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Title: Characterisation of a gene trap integration marking hepatic specification
Author: Watt, Alistair James
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1999
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Gene trapping in mouse embryonic stem cells has been used to identify and characterise the function of novel genes. Introduction of a gene trap vector into the genome results in the generation of a fusion between the lacZ reporter gene and the endogenous trapped gene. The consequences of this are predicted to be threefold: (i) expression of the reporter gene will be controlled by the promoter and enhancer elements of the endogenous gene and will therefore mirror endogenous gene expression; (ii) the generation of a fusion transcript allows endogenous gene sequence to be cloned by 5'RACE-PCR and (iii) the insertion of the gene trap vector can disrupt the function of the endogenous gene. This work describes the characterisation of one specific gene trap integration, 1114 that has not behaved entirely as predicted but has none-the-less identified an early marker of hepatic specification. The reporter activity profile associated with the 1114 gene trap integration is restricted to the definintive endoderm marking the ontogeny of the foetal liver. Reporter activity is observed as early as the 9 somite stage (8.0-8.5dpc) in endodermal cells of the foregut in the region destined to form the liver diverticulum and is restricted to the hepatic lineage until late gestation. Comparison of 1114 reporter activity with AFP identifies 1114 reporter activity as being the earliest, most specific marker of liver organogenesis identified to date. Breeding of the gene trap integration to homozygosity reveals no overt phenotype but its unique pattern of expression prompted us to clone the endogenous sequence. This has proven to be more complex than predicted as integration of the gene trap vector results in the production of two fusion transcripts. The most abundant (Group I) fusion transcript is ubiquitously expressed. No reporter activity is produced from this fusion transcript as gene trap vector splicing to this sequence places translation of the lacZ gene out-of-frame. The second, less abundant (Group II) fusion transcript is expressed exclusively in the liver during embryogenesis and is predicted to produce reporter activity. The cloning and sequencing of both the genomic sequence and the endogenous gene (gtar - gene trap nkyrin repeat) associated with the Group I and II fusion sequences has revealed that they represent different exons of gtar. Expression of the Group I and Group II sequences independent of the gene trap vector mimics that of the 1114 fusion transcripts. Furthermore, expression of these sequences in 1114 homozygous tissues indicates that the insertion of the gene trap vector has failed to disrupt the expression of gtar. Expression of the liver specific exon of gtar is postulated to be a consequence of either a separate promoter immediately upstream of the exon or alternative splicing.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available