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Title: Gene targeting in mouse embryonal stem cells
Author: Pells, Stephen Christopher
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1995
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This thesis is concerned with 'Hit and Run' gene targeting in ES cells, a method designed with the objective of introducing small mutations into the genome. The procedure relies on the integration of a vector carrying the desired mutation, and positive and negative selectable markers. After isolation of an event which has introduced the vector into the target locus by positive selection and genomic screening, a second selection performed upon the negative selectable marker is carried out. This should select for clones which have undergone an intra-chromosomal recombination event which removes the vector sequences and restores the locus. Some of the reverted clones obtained will have retained the mutation. The hit and run targeting technique relies on a high degree of fidelity in the homologous recombination process, to avoid the introduction of mutations other than the desired mutation into the target locus. However, relatively little is known about the fidelity of gene targeting homologous recombination events. Part of this thesis examines this question in a model gene, the hprtb-m3 null mutant locus. This allele is useful for studying gene targeting reactions because selections either for or against gene activity may be simple achieved an in vitro chemical selection. The hprtb-m3 gene was targeted with an insertion-type vector designed to restore hprt gene function which also incorporated the positive selectable marker neo. The neo gene was initially used to select stably-transfected clones. The clones were then studied genotypically and phenotypically to seek events where apparently correct homologous recombination events at the hprt locus failed to restore gene function and thus suggested errors in the targeting process. The data indicate that the rate of error in recombination must be low.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available