Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.653416
Title: Gene targeting in the mouse : introducing specific mutations associated with cystic fibrosis
Author: Kimber, Wendy Louise
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1995
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Abstract:
This thesis describes an attempt to create mouse models for CF bearing precise, CF-associated mutations as the only alteration to the murine Cftr gene, through use of the 'Hit and Run' gene targeting technique in mouse embryonal stem (ES) cells. This involves an initial targeting step in which an insertional targeting vector bearing the mutation to be introduced integrates through homologous recombination into Cftr. A negative selection then follows, enriching for those cells which have subsequently excised the vector and either reverted to the wild type genotype, or been converted to the desired genotype of the introduced mutation in the correct location as the only alteration to the gene. Vectors incorporating CF-associated mutations were introduced into ES cells and found to target Cftr at a higher frequency than had been previously reported for this region of the murine CF gene. Selection against the integrated vector in these targeted 'hit' clones was conducted with the unexpected result of 100% of resistant clones retaining the vector and the selection cassette. Close examination of these clones discovered no gross rearrangements, but a failure of methylation-sensitive restriction enzymes to cut within this region which could be induced or removed by growing the clone in negative or positive selection respectively. Further investigation of this phenomenon led to the conclusion that expression of the selection cassette was being modulated by methylation, and loss of the negative selection gene expression by this mechanism was occurring far more frequently than the desired loss through vector excision. The 'run' procedure was modified in view of this to reduce the high methylation-induced background, and clones were subsequently obtained which had survived the selection through vector excision.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.653416  DOI: Not available
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