The applicant proposes to utilize new approaches to target gene correction to mutations in the human rhodopsin gene associated with retinitis pigmentosa (RP). Site-directed correction of specific mutations will be accomplished by two oligonucleotide based approaches: triplex-forming oligonucleotides (TFOs) and RNA/DNA chimeric oligonucleotides. The feasibility of these approaches to gene targeting will be addressed at two levels: (1) in vitro experiments using TFOs to target both synthetic and plasmid-derived fragments of the rhodopsin gene; and (2) modified and unmodified TFOs and RNA/DNA chimeras will be tested in human cell lines that have been engineered to express normal and mutant versions of the rhodopsin gene from the native chromosomal locus. The major part of the proposed research will be to construct appropriate cell-lines to facilitate detection of a variety of mutations. This will be done by fusing the Green fluorescent Protein (GFP) to the rhodopsin gene. This construct will allow fluorescent-activated cell sorting to be used to collect altered cells.
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