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X. Liu, O.V. Bulgakov, B. Pawlyk, M. Adamian, T. Li; Phenotype Analyses in Mice With Targeted Disruption of Ush2A, a Mouse Model of Usher Syndrome Type IIA . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4589.
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To determine the subcellular localization of Ush2A protein and whether Ush2A deficiency leads to retinal degeneration and cochlear abnormalities.
The murine Ush2A coding sequence was determined by RT–PCR of retinal mRNA followed by DNA sequencing. Polyclonal antibodies were generated against recombinant fusion proteins corresponding to the N– and C–terminal regions of murine Ush2A. Mice lacking the Ush2A protein were generated by targeted gene disruption. WT and Ush2a–/– mice were analyzed by a variety of assays in both retinas and inner ears including electron microscopy, immunofluorescence, immunoblotting and electroretinographic (ERG) recording.
The murine Ush2A gene encodes a large polypeptide of 5193 amino acids, which shares 82% sequence identity with its human ortholog. Immunoblotting of retinal extracts confirmed that the major isoform of the Ush2A protein is the 600–KDa protein. Ush2A is expressed primarily in the apical region of the photoreceptor cell inner segment. Although the thicknesses of the outer nuclear layer (ONL), inner segment layer (IS) and outer segment layer (OS) of Ush2a–/– retinas appear normal in younger mice, the mutant retinas develop degeneration at older ages. In cochlear hair cells, Ush2A protein was found localized around the stereocilia. Scanning electron microscopy demonstrated loss of hair cells in Ush2a–/– mice.
Ush2A is a large extracellular matrix protein surrounding the connecting cilia of the photoreceptor cells as well as the stereocilia of cochlear hair cells, suggestive of a role in the maintenance of neurosensory ciliated cells. Ush2a–/– mice develop retinal degeneration and cochlear abnormalities, as found in human Ush2A patients.
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