Intraocular implantation of DNA-transfected retinal pigment epithelium cells: a new approach for analyzing molecular functions in the newt retinal regeneration

Abstract

Adult newts can regenerate their entire retinas, even after surgical removal of the neural retina (retinectomy), through transdifferentiation of the retinal pigment epithelium (RPE) cells. To develop a new experimental system for analyzing molecular functions during retinal regeneration of adult newts, we attempted to deliver a foreign gene into RPE cells of retina-less eye-cups in vitro. Here we used pCS2mt-GFP as a reporter construct, and selected Polyfect as a transfection reagent. DNA-transfection appeared to be restricted to the RPE cells of retina-less eye-cups and its efficiency was 0.1–0.2%. We tried to implant RPE-choroid tissue containing DNA-transfected RPE cells into the eye of a host animal. The tissue was placed into the posterior eye-chamber immediately after retinectomy so that the implanted RPE tissue was facing the cornea (i.e., normal orientation). The implant and host RPE regenerated one continuous hybrid neural retina. Ocular sections after 60 days of implantation showed that a small number of cells in the regenerating retina were intensely stained with an anti-GFP antibody. Some of those cells were believed to be retinal cells such as ganglion cells, amacrine cells and photoreceptors. The GFP-positive cells in the hybrid regenerating retina could represent clones derived from a single RPE cell. These results indicate that this experimental system could become useful in the study of adult newt retinal regeneration.