Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.662624
Title: Gene targeting and insertional mutagenesis in embryonic stem cells
Author: Sutherland, Helen F.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1992
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Abstract:
Mutations are important in the study of gene function. Until recently mouse mutants available for study were spontaneous mutations and those derived from chemical or X-ray mutagenesis. Transgenic technology brought about the creation of more mouse mutants through insertional mutagenesis by retroviral agent or microinjected recombinant DNA. The insert may be used as a tag to molecularly clone and characterise the mutated gene. With the advent of embryonic stem (ES) cell technology the number of mutations available for study should grow - both specific and random mutations. ES cells are pluripotent stem cells, that may be manipulated genetically in culture, and yet retain the ability to contribute to normal development and the germ line of a host blastocyst. Thus the phenotypic effects of the introduced mutation may be studied in vivo. Random mutations may be introduced into the ES cell genome by insertional mutagenesis by retroviral agent or by promoter or enhancer trap vectors. Gene targeting by homologous recombination may be used to introduce specific mutations. Hox-2.1 is a mouse homeobox-containing gene, belonging to the family of Hox genes. It has been cloned and mapped to the Hox-2 cluster on chromosome 11. Hox genes are thought to play important roles in mouse development. They may act as regulatory genes, controlling the expression of structural genes at different positions in the developing embryo. In order to study the function of Hox-2.1 in development it would be useful to have a mouse mutant for Hox-2.1. It was the aim of this project to produce a mouse mutant for Hox2.1. The first step towards this aim is the targeting/knock-out of the gene in ES cells. During this project various Hox-2.1 targeting constructs have been designed and built. Replacement vectors, incorporating a promoterless neo or employing the positive-negative selection strategy, were designed to enrich for targeting events. These and two insertion vectors were transfected separately into ES cells. Over 200 selected clones have been screened for disruption of the Hox2.1 locus. No homologous recombinants were identified. One clone, known as a 'pick-up' clone, was identified. It is thought that the targeting vector has found homology with the target sequence, picked up DNA from the locus (in this case 3' to sequence within the construct) and integrated elsewhere in the genome. An integration site, which the promoterless Hox2.1-neo targeting vector has integrated into twice, despite non-homology, has been characterised. It is suggested that this locus may be a site of frequent integration. The sequence flanking the construct was amplified by inverse PCR, cloned and sequenced. The properties which may make it highly targetable are discussed. It was shown to have weak promoter function.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.662624  DOI: Not available
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