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Author Notes:

Correspondence: Shannon E. Boye, shannon.boye@eye.ufl.edu.

P.D. Gamlin and S.E. Boye are joint senior authors.

We thank Enid Keyser, Debbie Whitten, Myra Rivers, Tracy Morris, Deidra Isbell, and Gregory Williams for their technical support.

Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Despite hosting the research topic together with one of the authors of this manuscript, the handling Editor states that the process met the standards of a fair and objective review.

Subjects:

Research Funding:

This work was supported by grants from the National Eye Institute, R01EY024280 (SEB), P30EY021721 (UF Vision Research Core grant), P30EY03039 (UAB Vision Research Core grant), R01EY025555 (PDG), Applied Genetics Technology Corporation (AGTC, Inc.) and an unrestricted grant from Research to Prevent Blindness.

Keywords:

  • Science & Technology
  • Life Sciences & Biomedicine
  • Neurosciences
  • Neurosciences & Neurology
  • macaque
  • photoreceptors (PRs)
  • retinal ganglion cells (RGCs)
  • adeno associated virus (AAV)
  • subretinal injection
  • lateral geniculate nuclei (LGN) injection
  • fluorescent activated cell sorting (FAGS)
  • ADENOASSOCIATED VIRUS VECTORS
  • LEBER CONGENITAL AMAUROSIS
  • MEDIATED GENE-THERAPY
  • PRIMATE RETINA
  • MACULAR PIGMENT
  • MONKEY RETINA
  • BIPOLAR CELLS
  • EXPRESSION
  • TRANSPLANTATION
  • PROMOTER

Novel Methodology for Creating Macaque Retinas with Sortable Photoreceptors and Ganglion Cells

Journal Title:

Frontiers in Neuroscience

Volume:

Volume 10, Number DEC

Publisher:

, Pages 551-551

Type of Work:

Article | Final Publisher PDF

Abstract:

Purpose: The ability to generate macaque retinas with sortable cell populations would be of great benefit to both basic and translational studies of the primate retina. The purpose of our study was therefore to develop methods to achieve this goal by selectively labeling, in life, photoreceptors (PRs) and retinal ganglion cells (RGCs) with separate fluorescent markers. Methods: Labeling of macaque (Macaca fascicularis) PRs and RGCs was accomplished by subretinal delivery of AAV5-hGRK1-GFP, and retrograde transport of micro-ruby™ from the lateral geniculate nucleus, respectively. Retinas were anatomically separated into different regions. Dissociation conditions were optimized, and cells from each region underwent fluorescent activated cell sorting (FACS). Expression of retinal cell type- specific genes was assessed by quantitative real-time PCR to characterize isolated cell populations. Results: We show that macaque PRs and RGCs can be simultaneously labeled in-life and enriched populations isolated by FACS. Recovery from different retinal regions indicated efficient isolation/enrichment for PRs and RGCs, with the macula being particularly amendable to this technique. Conclusions: The methods and materials presented here allow for the identification of novel reagents designed to target RGCs and/or photoreceptors in a species that is phylogenetically and anatomically similar to human. These techniques will enable screening of intravitreally-delivered AAV capsid libraries for variants with increased tropism for PRs and/or RGCs and the evaluation of vector tropism and/or cellular promoter activity of gene therapy vectors in a clinically relevant species.

Copyright information:

© 2016 Choudhury, Strang, Alexander, Scalabrino, Lynch Hill, Kasuga, Witherspoon, Boye, Gamlin and Boye.

This is an Open Access work distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).
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