Retroviral-Medicated Transfer of Suppressor tRNA Genes Into Human Cells
Breithaupt, Barbara Janine
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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.
x, 51 leaves. Advisor: Charisse M. Buising.