A method for isolation of cone photoreceptors from adult zebrafish retinae

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Title: A method for isolation of cone photoreceptors from adult zebrafish retinae
Authors: Glaviano, AntoninoSmith, Andrew J.Blanco-Fernandez, AlfonsoMcLoughlin, SarahCederlund, Maria L.Heffernan, TheresaSapetto-Rebow, BeataAlvarez, YolandaYin, JunKennedy, Breandán
Permanent link: http://hdl.handle.net/10197/8345
Date: 7-Nov-2016
Online since: 2017-02-15T12:33:33Z
Abstract: Background: Cone photoreceptors are specialised sensory retinal neurons responsible for photopic vision, colour perception and visual acuity. Retinal degenerative diseases are a heterogeneous group of eye diseases in which the most severe vision loss typically arises from cone photoreceptor dysfunction or degeneration. Establishing a method to purify cone photoreceptors from retinal tissue can accelerate the identification of key molecular determinants that underlie cone photoreceptor development, survival and function. The work herein describes a new method to purify enhanced green fluorescent protein (EGFP)-labelled cone photoreceptors from adult retina of Tg(3.2gnat2:EGFP) zebrafish. Results: Methods for dissecting adult zebrafish retinae, cell dissociation, cell sorting, RNA isolation and RNA quality control were optimised. The dissociation protocol, carried out with ~30 retinae from adult zebrafish, yielded approximately 6 × 106 cells. Flow cytometry cell sorting subsequently distinguished 1 × 106 EGFP+ cells and 4 × 106 EGFP− cells. Electropherograms confirmed downstream isolation of high-quality RNA with RNA integrity number (RIN) >7.6 and RNA concentration >5.7 ng/µl obtained from both populations. Reverse Transcriptase-PCR confirmed that the EGFP-positive cell populations express known genetic markers of cone photoreceptors that were not expressed in the EGFP-negative cell population whereas a rod opsin amplicon was only detected in the EGFP-negative retinal cell population. Conclusions: This work describes a valuable adult zebrafish cone photoreceptor isolation methodology enabling future identification of cone photoreceptor-enriched genes, proteins and signalling networks responsible for their development, survival and function. In addition, this advancement facilitates the identification of novel candidate genes for inherited human blindness.
Funding Details: Bright Focus Foundation
Macular Degeneration Grant
Welcome Trust
National Institutes of Health
Type of material: Journal Article
Publisher: BioMed Central
Journal: BMC Neuroscience
Volume: 17
Issue: 71
Copyright (published version): 2016 the Authors
Keywords: Cone photoreceptorsFlow cytometryCell sortingRNAZebrafish
DOI: 10.1186/s12868-016-0307-2
Language: en
Status of Item: Peer reviewed
This item is made available under a Creative Commons License: https://creativecommons.org/licenses/by-nc-nd/3.0/ie/
Appears in Collections:Conway Institute Research Collection
Biomolecular and Biomedical Science Research Collection

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