Molecular analysis of gene targeting mechanisms in mammalian cells

dc.contributor.advisorBaker, Mark D.
dc.contributor.authorNg, Philip of Molecular Biology and Geneticsen_US of Guelphen_US of Philosophyen_US
dc.description.abstractA two step gene targeting procedure for introducing planned genetic alterations into the hybridoma immunoglobulin [mu] locus in the absence of other extraneous modifications is described. The insertion vector contained the desired alteration within the region of homology to the target locus. A 'neo' gene was included in the vector backbone to facilitate isolation of G418 R primary recombinants that had undergone the first step of the procedure, vector integration by homologous recombination resulting in a duplication of the region of homology at the recombinant locus. The efficiency of the first step was improved 15- to 20-fold by using an enhancerless 'neo ' gene. The second step involved excision of the integrated vector by intrachromosomal homologous recombination between the duplicated region of homology. The HSV-1tk gene was included in the vector backbone to facilitate isolation of secondary recombinants. Vector excision was efficient, generating secondary recombinants at a rate of <math> <f> ~</f> </math>10-3/cell generation. As a result, the structure of the target locus was restored with the desired genetic alteration present in an expected proportion of the secondary recombinants. The mechanism of single copy targeted vector integration was studied by modifying the vector to contain six diagnostic restriction enzyme markers within the region of homology and was found to be consistent with the double-strand-break repair (DSBR) model of homologous recombination; the vector-borne marker proximal to the double-strand-break (DSB) is frequently lost and replaced by the corresponding chromosomal marker; formation of heteroduplex DNA (hDNA) was frequently associated with gene targeting and in most cases began about 645 bp from the DSB and could encompass a distance of at least 1469 bp, hDNA was efficiently repaired prior to replication, predominately by a long-patch mismatch repair mechanism. The mechanism by which targeted recombinants bearing >1 copy of the vector integrated at the target locus was investigated. Analysis of these recombinants suggested that they were generated by two DSBR events, each involving a single copy of the vector. The results also suggested that gene targeting and random vector integration were not directly competitive. Taken together, these results were interpreted to mean that a proportion of the recipient cells is in a state amenable to gene targeting.en_US
dc.publisherUniversity of Guelphen_US
dc.rights.licenseAll items in the Atrium are protected by copyright with all rights reserved unless otherwise indicated.
dc.subjectmammalian cellsen_US
dc.subjectmolecular analysisen_US
dc.subjectgene targetingen_US
dc.subjectgenetic alterationsen_US
dc.subjecthybridoma immunoglobulin locusen_US
dc.titleMolecular analysis of gene targeting mechanisms in mammalian cellsen_US


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