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M Natalia Vergara, Christian Gutierrez, M Valeria Canto-Soler; New Ex-Ovo Electroporation Technique Offers High Transfection Efficiency for Primary Retinal Cell Cultures. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1121.
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In vitro culture systems not only complement in vivo studies to further our understanding of retinal cell biology, but are also powerful tools for drug development applications and the identification of factors that promote cell survival and differentiation. For these and other purposes, the manipulation of gene expression in primary retinal cell cultures is of critical importance, yet achieving appropriate transfection efficiencies has remained challenging. The purpose of this work was to develop an ex-ovo electroporation technique that would allow transfection of chick retinal cells for primary cultures with higher efficiency than other currently available protocols.
Embryonic chick eyes were enucleated, devoid of RPE and sclera, and placed in an electroporation chamber filled with a plasmid solution. Square-wave electroporation was applied and, after removing the lens and vitreous, retinas were dissociated and cultured following standard techniques. Different parameters including voltage and number of pulses, plasmid concentration and location in relation to the retina, electrode type, and embryonic stage, were tested in order to optimize the electroporation conditions. Plasmids encoding GFP or Pax6-IRES-GFP under the control of a CAG promoter, were used. Transfection efficiency, cell viability and cell differentiation were analyzed with a Cellomics ArrayScan at 1 and 4 days in culture. Pax6 expression was analyzed by immunohistochemistry and Western blot.
The best results were obtained by using custom-made electrodes devised in our laboratory. The optimized protocol delivered transfection efficiencies in the order of 20-30% (a significant improvement over the 5-8% transfection efficiency obtained with other methods). Cell survival and cell-type ratios within the culture were not significantly affected by the procedure, and both photoreceptors and non-photoreceptor neurons were similarly transfected. A Pax6 encoding plasmid was electroporated as an example of overexpression of a biologically significant gene, resulting in Pax6 protein up-regulation and a concomitant decrease in photoreceptor cell differentiation.
Our goal was to provide the eye research community with an effective and efficient tool to manipulate gene expression in retinal cells in culture. This protocol offers a significant improvement over other currently available techniques, and we expect that it will prove useful to researchers working with this system.
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