A method for isolation of cone photoreceptors from adult zebrafish retinae.
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 × 106cells. Flow cytometry cell sorting subsequently distinguished 1 × 106EGFP+cells and 4 × 106EGFP-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.
UCD School of Biomolecular & Biomedical Science, UCD Conway Institute, University College Dublin, Dublin, D04 V1W8, Ireland.
Antonino
A
Glaviano
UCD School of Biomolecular & Biomedical Science, UCD Conway Institute, University College Dublin, Dublin, D04 V1W8, Ireland.
Andrew J
AJ
Smith
UCD School of Biomolecular & Biomedical Science, UCD Conway Institute, University College Dublin, Dublin, D04 V1W8, Ireland.
Alfonso
A
Blanco
UCD School of Biomolecular & Biomedical Science, UCD Conway Institute, University College Dublin, Dublin, D04 V1W8, Ireland.
Sarah
S
McLoughlin
UCD School of Biomolecular & Biomedical Science, UCD Conway Institute, University College Dublin, Dublin, D04 V1W8, Ireland.
Maria L
ML
Cederlund
UCD School of Biomolecular & Biomedical Science, UCD Conway Institute, University College Dublin, Dublin, D04 V1W8, Ireland.
Theresa
T
Heffernan
UCD School of Biomolecular & Biomedical Science, UCD Conway Institute, University College Dublin, Dublin, D04 V1W8, Ireland.
Beata
B
Sapetto-Rebow
UCD School of Biomolecular & Biomedical Science, UCD Conway Institute, University College Dublin, Dublin, D04 V1W8, Ireland.
Yolanda
Y
Alvarez
UCD School of Biomolecular & Biomedical Science, UCD Conway Institute, University College Dublin, Dublin, D04 V1W8, Ireland.
Jun
J
Yin
UCD School of Biomolecular & Biomedical Science, UCD Conway Institute, University College Dublin, Dublin, D04 V1W8, Ireland. brendan.kennedy@ucd.ie.
Breandán N
BN
Kennedy
D000818:Animals; D030801:Animals, Genetically Modified; D004210:Dissection / Q000379:methods; D005434:Flow Cytometry / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D017949:Retinal Cone Photoreceptor Cells / Q000166:cytology* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015027:Zebrafish
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D012313:RNA
Cell sorting; Cone photoreceptors; Flow cytometry; RNA; Zebrafish