| Autor: |
Choudhury S; Department of Ophthalmology, University of Florida Gainesville, FL, USA., Strang CE; Department of Psychology, University of Alabama at Birmingham Birmingham, AL, USA., Alexander JJ; Department of Human Genetics, Emory University Atlanta, GA, USA., Scalabrino ML; Department of Ophthalmology, University of Florida Gainesville, FL, USA., Lynch Hill J; Department of Ophthalmology, University of Alabama at Birmingham Birmingham, AL, USA., Kasuga DT; Department of Ophthalmology, University of Alabama at Birmingham Birmingham, AL, USA., Witherspoon CD; Department of Ophthalmology, University of Alabama at Birmingham Birmingham, AL, USA., Boye SL; Department of Ophthalmology, University of Florida Gainesville, FL, USA., Gamlin PD; Department of Human Genetics, Emory University Atlanta, GA, USA., Boye SE; Department of Ophthalmology, University of Florida Gainesville, FL, USA. |
| Abstrakt: |
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. |
