Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.641314
Title: Relationship between transgene copy number and variegated expression in mice
Author: Barkess, Grainne
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2001
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Abstract:
To test the hypothesis that high copy number arrays can be responsible for variegation, site-specific recombination was employed to reduce the copy number at the same genome location, allowing a direct comparison of different copy number arrays at the same integration site. A BLG-loxP transgene was microinjected to produce transgenic mice. High copy founders were used to establish lines and their expression profiles were analysed using in situ hybridisation, Northern blots, and milk protein composition. Two out of five lines showed variegated expression with discreet patches of cells expressing within the mammary gland. The variegating lines and one uniform line were then bred to a line of mice expressing a BLG-Cre recombinase transgene in the mammary gland. The double transgenic animals were analysed for a mammary-specific reduction in copy number and their expression patterns were also studied. In all three lines, after the reduction of copy number there had been a reduction in the number of cells in the mammary gland that expressed the BLG transgene. This was contradictory to the hypothesis that suggested that the variegation should be relieved by a reduction in copy number. The same BLG-loxP lines were then microinjected with a PGK-Cre recombinase construct to produce a reduction of copy number early in development. Microinjected animals were analysed for reduced copy arrays, of which only one line showed evidence. These animals were bred to establish lines with reduced copy number arrays throughout the animal. Their expression profile was analysed as before. Animals that had a reduction to one copy showed no expression, while animals that showed a reduction to two copies showed limited patches of expression. This result mirrored that of the mammary-specific reduction. BLG transgene may require a buffer zone provided by high copy arrays to allow some transgenes within the array to escape genome effects. When the copy number is reduced, the percentage of cells that can escape the silencing is also reduced. It is therefore clear that in some cases, transgenes may only efficiently express as a multicopy array.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.641314  DOI: Not available
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