Gene Transfer into Neurons of Organotypic Retina Cultures by Electroporation and Ballistic Transfection

Abstract

Purpose:: The highly specialized neurons in the network of the vertebrate retina survive and maintain their function only in the connection to their neighbouring and co functioning retinal cells. Because of this essential network, retinal cells, especially photoreceptor cells, can not be studied in primary cell cultures. For analyses of protein functions in retinal cells the organotypic retina culture provides an accessible ex vivo system, devoid of animal experiments.

Methods:: Retinas of maturating mice (PN8-PN19) were cultured and cell death was subsequently analyzed. Cultured retinas were transfected with eGFP constructs either by electroporation or ballistic transfection (Gene Gun^TM). The expression of eGFP fusion proteins in retinal cells were analyzed by fluorescence microscopy in whole mounts and cryosections. Transfection efficiencies were assessed for different ages of the explants as well as for different transfection methods. Furthermore, transfected cells were identified by co-labelling with antibodies against cell type specific marker molecules.

Results:: Retina cultures transfected via electroporation showed a higher amount of eGFP fusion protein expressing cells, especially in younger stages. In these younger explants (PN8), transfected cells were localized in the outer nuclear layer and in the inner nuclear layer, where as in the adult explants only cells of the retinal pigment epithelium could be transfected successfully. In contrast, gene transfers applied by the Gene Gun^TM method showed lower efficiency of transfection, but did deliver gene constructs also to cells of the inner retina. Ballistic transfection experiments also revealed that the transfection of specific retinal cell types correlated with the pressures of DNA gold practical acceleration applied.

Conclusions:: The organotypic retina culture proofed as vital and suitable system for gene transfers to different cell types of the retina ex vivo. The electroporation method is appropriate for transfection of retinal cells in the outer and inner most layers in juvenile retinas. By application of the Gene Gun^TM, fewer cells were transfected, but DNA constructs were successfully delivered to all retinal cell types, including cells of the inner retina which are commonly unreachable for external applied transfection reagents and gene constructs.

Keywords: retinal culture • gene transfer/gene therapy • cell survival