A method for isolation of cone photoreceptors from adult zebrafish retinae

BMC Neuroscience

Methodology Article

Yolanda Alvarez¹  Alfonso Blanco¹  Sarah McLoughlin¹  Beata Sapetto-Rebow¹  Jun Yin¹  Andrew J. Smith¹  Theresa Heffernan¹  Antonino Glaviano¹  Maria L. Cederlund¹  Breandán N. Kennedy¹ 

[1] UCD School of Biomolecular & Biomedical Science, UCD Conway Institute, University College Dublin, D04 V1W8, Dublin, Ireland;

关键词: Cone photoreceptors;    Flow cytometry;    Cell sorting;    RNA;    Zebrafish;   

DOI  :  10.1186/s12868-016-0307-2

 received in 2016-06-22, accepted in 2016-10-31,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundCone 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.ResultsMethods 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.ConclusionsThis 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.

【 授权许可】

CC BY   
© The Author(s) 2016