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Vincent T. Ciavatta, Staci A. Padove, Jeffrey H. Boatright, John M. Nickerson; Mouse Retina Has Oligonucleotide-Induced Gene Repair Activity. Invest. Ophthalmol. Vis. Sci. 2005;46(7):2291-2299. doi: 10.1167/iovs.04-1220.
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purpose. To test the ability of murine retinal nuclear extracts to support in vitro oligonucleotide-mediated gene repair (OMGR)—a prerequisite to repairing endogenous gene lesions underlying inherited diseases of the neurosensory retina.
methods. An in vitro reaction assayed whether retinal extract and oligonucleotides could correct a mutation in an antibiotic (tetracycline) resistance gene in a plasmid. The in vitro gene repair reaction combined plasmid, repair oligonucleotide, and nuclear extract. Site-specific repair of the tetracycline gene point mutation was revealed in a bacterial readout system in which plasmid from the in vitro reaction was recovered and electroporated into Escherichia coli. Colony growth on tetracycline indicated repair of the point mutation. To confirm site-specific nucleotide repair, plasmids were sequenced or subjected to restriction fragment length polymorphism (RFLP) analysis.
results. To quantify repair incidence, tetracycline-resistant colonies were normalized to ampicillin-resistant colonies. A repair oligonucleotide composed of RNA and DNA that contains one mismatched base relative to the target DNA induced an estimated 1 in 104 plasmids to be converted to wild type. If the extract was eliminated or boiled before reaction, no tetracycline-resistant colonies grew. Repair incidence increased with the concentration of retinal nuclear extract and oligonucleotide. Creating single-strand breaks in the plasmid caused a twofold increase in repair incidence.
conclusions. These in vitro assay data suggest that murine retina nuclei contain all the DNA repair factors necessary for OMGR, a finding that is prerequisite to attempting endogenous gene repair in mouse retina.
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