Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.645113
Title: Enhancing gene expression by using chromosomal sequences that flank an efficiently expressed transgenic locus
Author: Cranston, Anona Jane
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1996
Availability of Full Text:
Access from EThOS:
Full text unavailable from EThOS. Please try the link below.
Access from Institution:
Abstract:
The AATB transgene comprising 4.2 kb of the ovine BLG promoter sequences linked to human α1-antitrypsin (hAAT) genomic sequences gave high levels of hAAT expression in the mouse mammary. One line in particular (AATB 46.2) expressed hAAT at 6-8 mg/ml. This line was reported to have a low copy number loci, that could have been accommodated in a single cosmid vector. The aim of this work was to investigate the role of the flanking murine sequences in the high expression of the AATB transgene for this line. This thesis details the restriction characterisation of the AATB 46.2 genomic locus and the cloning of most (if not all) of the locus in several cosmids. A total of 4.6 kb of the 5' flanking murine sequences and 5 kb of the 3' flanking murine DNA were successfully isolated in separate cosmid clones. Comparison of the clones with genomic DNA demonstrated the locus had been cloned without rearrangement. Two secondary transgenes TAB and TA were constructed from these clones. TAB comprised the cloned 5' and 3' murine sequences flanking a single AATB transgene, while TA comprised the AATB transgene with only the 3' murine DNA from the AATB 46.2 locus. Transgenic mice were generated using these constructs. Expression of hAAT in the TAB transgenic mice was found to be improved compared with the AATB transgenic mice. Also the frequency of expression of the human protein by transgenic mice was increased. Ten of eleven transgenic founders/lines expressed the transgene at greater than 40μg/ml compared with eleven of twenty four for AATB. The levels hAAT expression in the secondary transgenic mice were generally very high and appeared to correlate with transgene copy number. By contrast expression of hAAT was not observed in any of the TA transgenic lines. The presence of either the 5' murine DNA alone or both the 5' and 3' murine DNA, may be required to bring about the improved expression profile of the biomedical transgene.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.645113  DOI: Not available
Share: