The long term goal of this research is to overexpress a mutant form of COII and create a dominant negative mutation in COX, transgenic mice containing the dominant negative mutation could be used to investigate the effects of the respiratory chain deficiencies which are characteristic of mitochondrial myopathies on mitochondrial biogenesis and gene expression. This thesis reports an attempt to express rat COII from a nuclear transgene. Using PCR strategies, the mitochondrial matrix targeting sequence from rat ornithine carbamyltransferase, was attached to a version of the rat COII gene which had been recoded into the universal genetic code. The transgene was cloned into the pLXSN retroviral vector and GP+E-86 ecotropic packaging cells were transfected with the construct. Infectious retrovirus particles were collected and used to infect C2C12 mouse myoblast and NIH3T3 mouse fibroblast cell lines. Expression of mRNA from the transgene was assessed in both cell lines using an RT-PCR strategy. For each cell type, protein expression from the transgene was assessed in the two clones showing the highest levels of mRNA expression. COII protein levels were determined by Western blot analysis using peptide-specific polyclonal antibodies directed to the C-terminus of rat COII. The transgene was expressed in a rabbit reticulocyte lysate system in which transcription and translation are coupled. In vitro translation products were immunoprecipitated using the COII peptide-specific antibodies. (Abstract shortened by UMI.)
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.20958 |
| Date | January 1998 |
| Creators | Hartlen, Rebecca. |
| Contributors | Shoubridge, Eric (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 | Master of Science (Department of Neurology and Neurosurgery.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001630789, proquestno: MQ50783, Theses scanned by UMI/ProQuest. |
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