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Muriël Messchaert, Ralph Slijkerman, Erwin van Wijk, Rob W J Collin; Functional characterization of EYS using zebrafish as a model organism. Invest. Ophthalmol. Vis. Sci. 2017;58(8):606.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in eyes shut homolog (EYS), a gene predominantly expressed in the photoreceptor cells of the retina, are among the most frequent causes of autosomal recessive retinitis pigmentosa (RP), accounting for approximately 5-10% of all cases. The function of EYS in photoreceptor cells and the pathogenic mechanism underlying EYS-associated RP are still unclear. In this study we aim to unravel the function of EYS using zebrafish as a model organism.
We cloned and characterized eys in zebrafish, via RT-PCR analysis on zebrafish eyes cDNA and sequencing. Furthermore, we generated a stable eys knock-out zebrafish model by adopting CRISPR/Cas9 technology. RT-PCR was performed to evaluate the expression of eys mRNA in wild type and mutant zebrafish larvae. The effect of the absence of Eys on retinal structure and function will be studied using morphological analysis and functional assays.
RT-PCR on zebrafish eyes cDNA resulted in the identification of a transcript of 8598 base pairs consisting of 45 exons. This transcript is predicted to encode a 2866 amino acid long protein containing 38 EGF-like domains and five laminin A G-like domains. The encoded protein shows 32% sequence identity on the amino acid level compared to human EYS. Co-injection of Cas9 protein with a gRNA targeting eys exon 19 into zebrafish embryos introduced a five base pairs deletion (c.3371_3375delGGGAG, p.Gly1124Valfs*15). It is predicted to lead to a premature stop codon, resulting in a truncated protein. RT-PCR analysis showed the presence of mutant eys transcript in homozygous mutant zebrafish larvae.
We characterized eys in zebrafish, which shows high similarity to human EYS. Furthermore we were able to create a stable eys knock-out zebrafish line. Our work will serve as the basis for understanding the function of EYS, and to initiate pre-clinical therapeutic intervention studies.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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