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RANZCO Abstract Gene Editing in Zebrafish for functional characterisation of a PDE6B founder mutation causing autosomal recessive rod-cone retinal dystrophy in Māori, Presenter: Micah Rapata1 Co-authors: Lin Hou1, Verity Oliver1, Andrea Vincent1,2 Institutions: 1.Department of Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and Health Sciences, University of Auckland, New Zealand; 2. Eye Department, Greenlane Clinical Centre, Auckland District Health Board, Auckland, New Zealand. Presenter email: mrap925@auckland.ac.nz Type of presentation: Oral Purpose To create, and phenotype, a zebrafish model of a novel founder PDE6B mutation (c.2197G>C; p.Ala733Pro) in the Māori population causing rod-cone dystrophy, to enable high throughput screening of novel therapeutics targeting cyclic GMP. Methods Transient morpholino knockdown of PDE6B was performed on zebrafish embryos, with validation by RT-PCR. CRISPR gRNA-Cas9 complexes were injected into zebrafish embryos to create a stable PDE6B mutant. Phenotypic analysis of morpholino and CRISPR/Cas9- edited zebrafish was performed using light microscopy imaging, OCT, histology and cGMP expression by immunohistochemistry. Functional vision was assessed via the optokinetic response. Results RT-PCR confirmed temporary knockdown of PDE6B, while CRISPR/Cas9 mutants are currently awaiting validation. Gross morphological changes were observed in day 5 embryos, including decreased ocular pigmentation. In day 7 embryos, no structural differences on histology, nor reduction in the optokinetic response were seen. Conclusion Morpholino and CRISPR/Cas9 systems can successfully knockdown PDE6B gene function in a zebrafish retinal degeneration model, causing phenotypic and morphological changes in embryonic zebrafish retinas. No obvious effect on functional vision was observed with the morpholino model. Utilizing the CRISPR/Cas9 system to create a stable PDE6B zebrafish model will permit characterization of the degenerative process of the retina including assessment of cGMP levels, and allow therapeutic drug screening. |
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