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F. Chan, K. Sykoudis, A. K. Gross, I. M. Sandoval, B. Price, D. Mittelman, S. H. Min, W. W. Hauswirth, T. G. Wensel, J. H. Wilson; Rhodopsin Gene Correction in Rod Cells of Mice. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4613.
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Dominant mutations in the rhodopsin gene are a major cause of the blinding disease, autosomal dominant retinitis pigmentosa (ADRP). Effective methods for rhodopsin gene correction (or gene knockout) could serve both as the basis for treatment of ADRP, and as a paradigm for treating other dominant diseases. Detecting gene correction, especially in initial stages when success is likely to be rare, requires highly sensitive methods, ideally capable of identifying a single event in a retina. To this end, we have created several mouse models that have one mouse rhodopsin allele replaced by a modified human rhodopsin-GFP fusion gene, whose expression confers bright green fluorescence on the rod outer segment.
Homologous recombination (HR) in mouse embryonic stem cells was used to create knock-in mice. Two contain nonsense mutations, Q64Ter or Q344Ter, which are found in patients with ADRP. Another contains a duplication of exon 2, which leads to a frameshift and premature termination. The intron between the two copies of exon 2 contains a site for the rare-cutting restriction enzyme ISce-1. Adeno-associated virus (AAV) directing expression of ISce-1 under control of the rhodopsin promoter was injected into the subretinal space. Rod outer segments containing rhodopsin-GFP were detected by fluorescence microscopy of retinal whole mounts.
All of the knock-in alleles were successfully passed from chimeric mice through the germ line. No GFP fluorescence was detected in the untreated retinas of mice with alleles expressing the truncated rhodopsins. Injection of ISce-1-encoding AAV into the retinas of mice with the exon-2 duplication resulted in rod cells expressing rhodopsin-GFP in their outer segments.
Double strand breaks introduced by ISce-1 appear to have induced intra-chromosomal HR and gene repair in the exon-2 duplication allele. Rhodopsin-GFP knock-in mice provide a powerful and sensitive system for detecting gene repair events.
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