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V.T. Ciavatta, S.A. Padove, J.H. Boatright, J.M. Nickerson; Alternative RDO designs in gene repair assays with retinal nuclear extracts . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3483.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: We previously reported that nuclear extract from neural retina proved capable of supporting oligonucleotide–mediated gene repair in an in vitro assay (ARVO 2002, abstracts 2334, 2336). Seeking to enhance the rate of gene repair, we examined the effects of altered oligonucleotide (oligo) designs on repair rate. We also sought to compare the rate of gene repair supported by retinal extracts from young (PN21) versus old (>1 yr) mice, hypothesizing there would be no difference as endogenous gene repair mechanisms should be essential to cells from both retinas. Methods: Plasmid pBRTETs is a tetracycline–sensitive version of pBR322 in which a point mutation creates a premature stop codon in the tet gene. The original RDOTET1 repair oligo has a mismatch with both the template and non–template strands at the point mutation in pBRTETs. The oligo was altered to have either no mismatch (RDOTET3), or a single mismatch with the template strand (RDOTET2) or the non–template strand (RDOTET5). Retinal nuclear extracts were from C57/BL6, Balb/C, and C57/BL6 X Balb/C mice. Complete in vitro reactions, in which nuclear extracts were incubated with plasmid DNA and a repair oligo, and genetic readout in E. coli, were used to assess gene repair rate as the ratio of tet–resistant colonies to amp–resistant colonies. Results: Typical gene repair rate (tet–resistant colonies per million amp–resistant colonies) using RDOTET1 is about 4. Substituting RDOTET2 or RDOTET5 for RDOTET1 increased the repair rate to 40.2 +/– 28.5 (n=9) and 14.4 +/– 10.11 (n=6), respectively. RDOTET3 decreased the repair rate (0.0837 +/– 0.0765 (n=3)). Repair rates from the no–extract control reactions employing RDOTET2, RDOTET5, and RDOTET3 were 0.73 +/– 0.15 (n=3), 7.2 +/– 5.4 (n=6), and 0.0833 +/– 0.0723 (n=3), respectively. The complete repair reaction for RDOTET2 was higher than its no extract control at p </= 0.0016, whereas the complete reaction for RDOTET3 was indistinguishable from its no extract control. Initial results indicate that retinal nuclear extract from older mice results in higher repair than extract from weanlings. Conclusions: Single mismatch RDOs improved repair rates, as RDOTET2 markedly enhanced gene repair 10–fold over the original RDOTET1. Optimization of oligo design is necessary if oligonucleotide–mediated gene repair is to become a viable strategy. The in vitro gene repair assay is a facile method for evaluating effects of oligo design and extract source. More work is needed to confirm if mouse age affects gene repair rate.
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