PubAg

Main content area

Efficient mutagenesis of the rhodopsin gene in rod photoreceptor neurons in mice

Author:
Chan, Fung, Hauswirth, William W., Wensel, Theodore G., Wilson, John H.
Source:
Nucleic acids research 2011 v.39 no.14 pp. 5955-5966
ISSN:
0305-1048
Subject:
Dependoparvovirus, annealing, loci, humans, neurons, rhodopsin, mutagenesis, homologous recombination, mice, DNA repair, nucleic acids, exons
Abstract:
Dominant mutations in the rhodopsin gene, which is expressed in rod photoreceptor cells, are a major cause of the hereditary-blinding disease, autosomal dominant retinitis pigmentosa. Therapeutic strategies designed to edit such mutations will likely depend on the introduction of double-strand breaks and their subsequent repair by homologous recombination or non-homologous end joining. At present, the break repair capabilities of mature neurons, in general, and rod cells, in particular, are undefined. To detect break repair, we generated mice that carry a modified human rhodopsin-GFP fusion gene at the normal mouse rhodopsin locus. The rhodopsin-GFP gene carries tandem copies of exon 2, with an ISceI recognition site situated between them. An ISceI-induced break can be repaired either by non-homologous end joining or by recombination between the duplicated segments, generating a functional rhodopsin-GFP gene. We introduced breaks using recombinant adeno-associated virus to transduce the gene encoding ISceI nuclease. We found that virtually 100% of transduced rod cells were mutated at the ISceI site, with ~85% of the genomes altered by end joining and ~15% by the single-strand annealing pathway of homologous recombination. These studies establish that the genomes of terminally differentiated rod cells can be efficiently edited in living organisms.
Agid:
2378604