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C. Andrieu, F. Behar–Cohen, A.M. Faussat, J.P. Concordet; In vitro Correction of the rd1 Point Mutation in the ß–phosphodiesterase Gene by Specific Single–Stranded Oligonucleotides . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5198.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To establish a reproducible in vitro model for analyzing the factors influencing the frequency of rd1 mutation correction by generating 293 cells containing a single stable integrated copy of the mutated rod cGMP–phosphodiesterase (stop mutation) fused to GFP reporter cDNA (ß–PDErd1–GFP). Methods: Following electroporation of linearized pFRT/LacZeo plasmid, 293 cell clones having integrated pFRT/LacZeo were selected on Zeocin. The clones containing a single FRT site were identified by Southern blot analysis. Three of these clones were then transfected with pcDNA5/FRT/TO/ß–PDErd1–GFP and selected with the appropriate antibiotic, hygromycin. Southern blot analysis was used to confirm that these stable clones contained a single copy of the ß–PDErd1–GFP fusion construction. Phosphorothioate ODNs encoding the wild type ß–PDE allele targeting the transcribed or the non–transcribed DNA strand (respectively sense or antisense) were transfected in 293–ß–PDErd1–GFP using several transfecting agents and various conditions of transfection (number of candidate cells, amount of transfecting agent, amount of ODN, ratio of ODN / transfecting agent, volumes and duration for transfection). Correction of the rd1 stop codon mutation leads to the expression of GFP. The percentage of GFP–positive cells was quantified by flow cytometry. Experiments were repeated four times. Results: Our findings demonstrate that up to 2% gene correction is achieved in vitro on genomic rd1 mutation with specific phosphorothioate ODNs. Amounts of ODNs and transfection efficacy were crucial for gene repair in vitro. The best transfecting agent in 293 cells was Lipofectamin 2000. Both the sense and antisense specific ODNs induced the correction of the rd1 mutation but a higher correction was observed with the antisense phosphorothioate ODN (1.8–2.1%) compared to the sense ODN (0.5–1.5%). Conclusions: The rd1 mutation (stop codon on amino acid position 347) can be corrected by single stranded phosphorothioate ODNs when integrated in the genomic DNA of 293 cells. This in vitro model allows the study of gene repair mechanisms and the selection of optimal parameters to enhance gene correction for potential in vivo applications.
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