Retroviral-Medicated Transfer of Suppressor tRNA Genes Into Human Cells

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Show simple item record Breithaupt, Barbara Janine 2006-02-28T17:03:44Z 2006-02-28T17:03:44Z 2000-03
dc.identifier.other 2000 .B748
dc.description x, 51 leaves. Advisor: Charisse M. Buising. en
dc.description.abstract Nonsense mutations in protein genes have been associated with a variety of human diseases. A useful therapeutic approach to correct such mutations may be to introduce genes expressing suppressor tRNA that recognize the premature stop codon, insert the appropriate amino acid, and allow translation of a full-length, functional protein. Human opal arginine suppressor tRNA genes that encode the structural portion of the tRNA and about 15 bases from the 3' flanking sequence were synthetically constructed using oligonucleotides. Retroviral vectors were selected for delivery to human target cells in order to stably integrate the suppressor tRNA into the target cells' genome. Recombinant retroviral vectors were used to transfect retrovirus packaging cells and produce recombinant viral supernatants. Recombinant viral supernatants were then used to transduce the target cells. Expression of suppressor tRNA in both the retrovirus packaging cells and in the target cells was confirmed by slot blot analysis. To test the functional activity of the expressed suppressor tRNA, target cells expressing a nonsense mutated GFP reporter gene (Arg73opal mhRGFP) were transduced with the recombinant viral supernatant. Suppression of the opal nonsense mutation in hRGFP was demonstrated by restoration of hRGFP fluorescence. This study suggests that retroviral vectors may be used to deliver suppressor tRNA genes to human target cells. en
dc.format.extent 11551420 bytes
dc.format.mimetype application/pdf
dc.language.iso en_US
dc.publisher Drake University en
dc.relation.ispartofseries Drake University Theses, The College of Arts and Sciences;2000
dc.subject Recombinant proteins--Synthesis. en
dc.subject Gene therapy en
dc.subject Retroviruses. en
dc.title Retroviral-Medicated Transfer of Suppressor tRNA Genes Into Human Cells en
dc.type Thesis en

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