Lipid-mediated gene transfection into chick embryo retinal cells in ovo and in vitro

• Published in: Journal of neuroscience methods Vol. 104; no. 1; pp. 1 - 8
• Main Authors: Toy, Jeffrey, Bradford, Rebecca L, Adler, Ruben
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 15-12-2000; Elsevier Science
• Subjects: Animals; Biological and medical sciences; Cell Culture Techniques; Cells, Cultured; Chick Embryo; DNA - pharmacology; Eye and associated structures. Visual pathways and centers. Vision; Fundamental and applied biological sciences. Psychology; Gene expression; GenePORTER; Genes - physiology; Genes, Reporter - physiology; Green Fluorescent Proteins; Indicators and Reagents - metabolism; Lens; Lipofection; Liposomes - pharmacology; Luminescent Proteins - genetics; Neurons - cytology; Neurons - drug effects; Neurons - metabolism; Retina - drug effects; Retina - embryology; Retina - metabolism; Retina development; Retinal cell culture; Retroviridae - genetics; Retrovirus; Transfection; Vertebrates: nervous system and sense organs; Retina development; Retrovirus; Retinal cell culture; Transfection; Chick embryo; Lipofection; Lens; GenePORTER; Gene expression; Embryo; Retroviridae; Lipids; Retina; Eye; Virus; Visual system; Vertebrata; Reporter gene; Green fluorescent protein; Development; Chicken; Aves
• ISSN: 0165-0270; 1872-678X
• DOI: 10.1016/S0165-0270(00)00311-3

Several lipofection reagents were tested on chick embryo retinal cultures using green fluorescent protein (GFP) as a reporter gene; best results were obtained with the GenePORTER™ (GP) reagent, which yielded approximately 4.4% of the cells with intense GFP fluorescence. Cell survival and structural differentiation appeared normal, but one of the immunocytochemical markers studied (visinin) was less frequently observed in GP-treated cultures. When similar plasmid-GP mixtures were injected into chick embryo eyes in ovo, bright GFP-fluorescent cells were observed in different retinal layers, without detectable detrimental effects on retinal morphology. Particularly extensive reporter gene expression was obtained upon intraocular injection of GP plus naked DNA from a RCAS retrovirus, which resulted in the development of abundant radial columns of alkaline phosphatase-positive cells, separated by columns of negative cells. We conclude that lipid-based transfection offers a quick, simple and fairly innocuous means for gene delivery into proliferating and postmitotic retinal cells, in vitro as well as in the developing eye in ovo, and that transfection of naked retroviral DNA can lead to extensive expression of foreign genes by retinal cells, bypassing the time-consuming steps required for the generation of high-titer virion stocks.