ZFIN ID: ZDB-PUB-161110-24
A method for isolation of cone photoreceptors from adult zebrafish retinae
Glaviano, A., Smith, A.J., Blanco, A., McLoughlin, S., Cederlund, M.L., Heffernan, T., Sapetto-Rebow, B., Alvarez, Y., Yin, J., Kennedy, B.N.
Date: 2016
Source: BMC Neuroscience   17: 71 (Journal)
Registered Authors: Alvarez, Yolanda, Cederlund, Maria, Kennedy, Breandan N., McLoughlin, Sarah, Yin, Jun
Keywords: Cell sorting, Cone photoreceptors, Flow cytometry, RNA, Zebrafish
MeSH Terms:
  • Animals
  • Animals, Genetically Modified
  • Dissection/methods
  • Flow Cytometry/methods*
  • Green Fluorescent Proteins/genetics
  • Green Fluorescent Proteins/metabolism
  • RNA/metabolism
  • Retinal Cone Photoreceptor Cells/cytology*
  • Retinal Cone Photoreceptor Cells/metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Zebrafish*
PubMed: 27821066 Full text @ BMC Neurosci.
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ABSTRACT
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.
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.
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.
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