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Astra Dinculescu, Omar Akil, Susan Bolch, Frank M Dyka, William N Ruddick, Chiab Simpson, Lei Xu; A novel epitope-tagged Clarin-1 knock-in mouse with retinal dysfunction and profound congenital deafness. Invest. Ophthalmol. Vis. Sci. 2020;61(7):2446.
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© ARVO (1962-2015); The Authors (2016-present)
Usher syndrome type III (USH3) is a recessive inherited disorder caused by mutations in the Clarin-1 (CLRN1) gene, leading to progressive hearing loss and retinal degeneration. CLRN1 is expressed at low levels in sensory organs. Our recent work revealed that CLRN1 transcripts in adult mouse and human retinas are found in Müller glia and not photoreceptors (Xu et al, Journal of Pathology, 2019). In order to bypass the limitations in the availability of CLRN1-specific antibodies, we generated and characterized hemagglutinin (HA) epitope-tagged Clrn1 knock-in mice to facilitate CLRN1 protein detection.
N-HA and C-HA epitope-tagged Clrn1 knock-in mice were generated, in which the HA tag was inserted either in the beginning of the Clrn1 coding region, or right before its stop codon. Retinal function and hearing were assessed by electroretinography (ERG) and acoustic brainstem response (ABR) testing, respectively. Retinal pathology was evaluated with spectral-domain optical coherence tomography (OCT). Anti-HA antibodies were used in immunoblotting and immunofluorescence experiments.
We detected the presence of a characteristic diffuse HA-tagged CLRN1 protein band by Western blot analysis in retinal lysates from both N-HA and C-HA Clrn1 knock-in mice throughout development and adulthood. CLRN1 expression was below the detection limit of immunohistochemistry assays. The N-HA Clrn1 knock-in mice had normal vision and hearing. In contrast, homozygous C-HA Clrn1 knock-in mice displayed profound congenital hearing loss and progressive retinal dysfunction. Remarkably, the auditory phenotype in the homozygous C-HA Clrn1 knock-in mice is even more severe than in Clrn1 knockout (KO) or N48K knock-in mouse models, which display an early onset but progressive hearing loss. ERG analysis of retinal function showed that both a- and b-wave amplitudes were significantly decreased by 25% (P < 0.05) in 6-month-old homozygous C-HA Clrn1 knock-in mice compared to age-matched wild-type controls. Histological examination and OCT revealed no retinal abnormalities.
CLRN1 protein is expressed at comparable levels in both HA-tagged Clrn1 knock-in mouse retinas, as determined by Western blot assays. The placement of the HA-tag at the C-terminal end of CLRN1 interferes with its biological function, and increases the severity of the auditory phenotype relative to mouse models of USH3.
This is a 2020 ARVO Annual Meeting abstract.
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