Biodistribution of Rat Primary Cells transfected with SB100X transposon-mediated Pigment Epithelium-Derived Factor (PEDF) gene in Brown Norway rats after subretinal injection

Abstract

Purpose: We aimed to identify, localize and characterize the rat primary cells: retinal pigment epithelium cells (RPEs) and iris pigment epithelium cells (IPEs) transfected with pFAR4-CMV-Venus plasmid or pFAR4-CMV-pigment epithelium-derived factor (PEDF) plasmid with Sleeping Beauty (SB100X) transposon in the subretinal space of pigmented Brown Norway rats.

Methods: RPE and IPE rat primary cells (20,000 cells) were transfected with SB100X transposase with Venus or PEDF-Histidine gene encoding pFAR4 plasmids (SB100X-pFAR4-Venus/PEDF-His) (ratio 1:16 / 0.5 µg total DNA per transfection) using an electroporator (Neon Transfection System). We injected transfected cells in the subretinal space of healthy female Brown Norway rats (n=50) and then the animals were sacrificed at different time points (7, 15 30, 90 and 120 days postinjection). The eyes were enucleated and the flat mounts and retinal sections were labeled with nuclear marker (TOPRO-3) and visualized under the confocal microscope to observe the Venus transfected cells. Immunofluorescence for PEDF (monoclonal antibody, Chemicon, MAB1059) and Histidine (polyclonal antibody, Novus, NBP1-25939) antibodies were performed in flat mount retinas to analyze the expression in transfected primary cells after 21 and 28 days postinjection.

Results: We identified the Venus transfected primary cells in the area of the injection in cross retinal sections close located to the RPE. Engraftments of primary cells were observed in flat mounted retinas in all groups after different time points. Double immunofluorescence for PEDF and Histidine revealed that both molecules colocalized in primary cells injected subretinally, and PEDF expression was observed even in the long term samples analyzed.

Conclusions: The subretinal injection of RPE and IPE cells transfected with SB100X-pFAR4-Venus/PEDF-His is safe. We demonstrate in vivo in a rat model the presence of these cells in the target area and its ability to express PEDF over the time by using the SB100X transposon system technology.