In eukaryotes homologous recombination is the basis of such important biological events as meiotic recombination, generation of the antigen recognition molecule repertoire of the immune system and repair of DNA double strand breaks (DSBs). / Homologous recombination between an exogenous DNA sequence and a chromosomal sequence is termed "gene targeting". In the future, gene therapy will predictably become the treatment of choice for a number of inherited and acquired genetic diseases. The main obstacle to the application of gene targeting to gene therapy is the low frequency of homologous recombination in mammalian somatic cells. The ability to specifically increase the frequency of homologous recombination in mammalian cells could make gene targeting applicable to gene therapy of hereditary genetic disorders and cancer. / Here, we present the results of our studies in which we explored two approaches for transient increase of the frequency of homologous recombination in mammalian somatic cells: expression of the S. cerevisiae endo-exonuclease (EE) NUD1 gene, and inhibition of poly(ADP-ribose) polymerase (PARP) with 1,5-isoquinolinediol (ISQ). / We have demonstrated that transient expression of NUD1 in HeLa cells increased the resistance of the latter to ionizing radiation and cis-platin, two agents known to cause DNA DSBs, whereas it had no effect on cells' resistance to DNA-methylating agents. We have also shown that transient expression of NUD1 enhanced the frequency of extrachromosomal homologous recombination in mouse Ltk-cells. These results provide additional evidence for the involvement of EEs in homologous recombination in mammalian cells. They also demonstrate that overexpression of EEs, such as Nud1p, can potentially be used as a means of selectively increasing the frequency of homologous recombination in mammalian cells without a concomitant increase in the frequency of illegitimate recombination. / Our studies of PARP inhibition showed that treatment of cells with 0.622 mM ISQ results in an average 4.6-fold increase in the frequency of extrachromosomal homologous recombination in mouse Ltk-fibroblasts. We have also demonstrated that treatment of mouse Ltk-cells with ISQ leads to an increase of up to 8-fold in the absolute frequency of gene targeting of the stably integrated HSV tk gene. We believe that our results concerning the effect of ISQ on gene targeting may have potential application for the improvement of such technologies as the generation of genetic knock-outs and the ex vivo gene therapy. Our findings also appear to solve the conflict in the previously reported results that PARP inhibition stimulates chromosomal recombination but not extrachromosomal homologous recombination or gene targeting.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.37600 |
| Date | January 1999 |
| Creators | Semionov, Alexandre. |
| Contributors | Cournoyer, Denis (advisor), Chow, Terry (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: 001754123, proquestno: NQ64665, Theses scanned by UMI/ProQuest. |
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