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Title: Visualising Ren-1d gene expression in transgenic mice by using in vivo modified genomic clones
Author: Payne, Catherine M.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1999
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The As4.1 cell line expresses its endogenous renin gene, and constitutively secretes prorenin into the supernatant, but can no longer be stimulated to release active renin. Whilst primary JGC cultures are only 80% pure, and lose their renin activity within 3 weeks of culture. Therefore there is a requirement for a cell line which an be stimulated to secrete renin, and which is as close to a pure primary JGC culture as possible. This thesis describes the in vivo modification of genomic clones spanning the renin locus. P1251, and the bacterial artificial chromosome (BAC), N10, were used for intermolecular recombination, to introduce the β-Geo reporter gene, fused to an internal ribosomal entry site, into Ren-1d. Initially, recombination between the P1 and a linear fragment, in a recombination proficient E. coli strain, produced many false positive clones, indicating a requirement for negative selection. The BAC targeting strategy, which utilises a temperature sensitive suicide vector, was successfully used to introduce the reporter specifically into Ren-1d, with no recombination observed in Ren-2, or any other region of the P1 or BAC. Intermolecular recombination, using the suicide vector, was also used to introduce site specific modifications into the Ren-1d gene. Numerous co-integrations were obtained, although to date, none of the resolved clones analysed have contained the mutated sequences, suggesting a different screening strategy is necessary. The transgenic mouse, TGM(N10Rn1βGeo), is the first transgenic animal to express a reporter gene in renin expressing tissues. β-Geo is expressed throughout development and in the adult, and will facilitate the isolation of pure primary JGCs.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available