Ectopic gene targeting is an alternative outcome of the gene targeting process in which the targeting vector acquires sequences from the genomic target but proceeds to integrate elsewhere in the genome. To explore this mechanism of recombination an assay was developed, designated as Recombination Access Mapping (RAM), that uses ectopic gene targeting coupled with two color FISH analysis. Using RAM, the integration sites of the gene targeting vector with respect to the target locus were determined for ectopic gene targeting in a murine fibroblast cell line (LTA). The distribution of integration sites was found to be bimodal during ectopic gene targeting, being either within 2--3 Mb of the target or on distinct chromosomes from the chromosome carrying the target locus. Both inter and intrachromosomal sites appeared to be equally accessible to the targeting vector with site specific variations. In contrast, illegitimate integration of a transgene was found to occur randomly in the LTA cell line. Interestingly, interphase analysis indicated that vector sequences which had integrated ectopically in other chromosomes than the target co-localized with the target locus at a significant frequency as compared to random unlinked loci. Mechanistically, the homologous recombination event that produces the extrachromosomal recombinant DNA molecule, following ectopic gene targeting or interplasmid recombination, was demonstrated to potentiate or commit the recombinant DNA to integration in the genome at the site of a double-strand break (DSB). This data suggests that extrachromosomal and chromosomal DSB repair are coupled, and therefore the integration site of the transgene during ectopic gene targeting is most likely a DSB in close proximity spatially to the target locus. The conclusion of this thesis is that RAM can be used to determine which chromosomal domains within the genome are accessible to a given genetic locus. Thus, RAM presents a new paradigm for the analysis of DN
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.34942 |
| Date | January 1998 |
| Creators | Dellaire, Graham. |
| Contributors | Chartrand, Pierre (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Division of Experimental Medicine.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001635477, proquestno: NQ44404, Theses scanned by UMI/ProQuest. |
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