Gene targeting is a process of recombinational exchange between a foreign molecule of DNA and its homologous chromosomal counterpart whereby the cellular machinery involved in homologous recombination accomplishes sequence exchange. The widespread use of gene targeting as a basic research and/or therapeutic tool has been precluded by its inherent inefficiency. Although many studies have reported improved gene targeting efficacy via manipulation of homologous recombination-associated proteins, reports examining the underlying molecular mechanisms of gene targeting are scant. Improvement in our understanding of these molecular mechanisms will undoubtedly lead to advancements in the study of genetic disease, and perhaps to the development of viable human gene therapy. To this end, the following is a report of the development and preliminary assessment of a model system for the study of the molecular mechanisms of gene targeting. Specifically, this system entailed the investigation of recombinational exchange between isogenic, non-functional copies of HSV-tk contained in a targeting plasmid and a stably integrated target plasmid, in Ltk-cells. Results showed an absolute random integration frequency of our targeting construct in the range of 10-4---a value ten-fold lower than classically associated with random integration. Also, linearization of our targeting plasmid outside the region of homology to our stably integrated chromosomal target appeared to be associated with an undetectable level of targeting. Similarly, non-homology---in the form of three non-silent point mutations in our target sequence---appeared to impede gene targeting. These initial results suggest that the design of this model system needs to be revised to allow proper investigation of the molecular mechanisms underlying gene targeting.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.84103 |
| Date | January 2006 |
| Creators | Paiement, Jean-Pierre |
| 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 | Master of Science (Division of Experimental Medicine.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 002588270, proquestno: AAIMR32641, Theses scanned by UMI/ProQuest. |
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