Ex vivo electroporation of retinal cells: a novel, high efficiency method for functional studies in primary retinal cultures.
Primary retinal cultures constitute valuable tools not only for basic research on retinal cell development and physiology, but also for the identification of factors or drugs that promote cell survival and differentiation. In order to take full advantage of the benefits of this system it is imperative to develop efficient and reliable techniques for the manipulation of gene expression. However, achieving appropriate transfection efficiencies in these cultures has remained challenging. The purpose of this work was to develop and optimize a technique that would allow the transfection of chick retinal cells with high efficiency and reproducibility for multiple applications. We developed an ex vivo electroporation method applied to dissociated retinal cell cultures that offers a significant improvement over other currently available transfection techniques, increasing efficiency by five-fold. In this method, eyes were enucleated, devoid of RPE, and electroporated with GFP-encoding plasmids using custom-made electrodes. Electroporated retinas were then dissociated into single cells and plated in low density conditions, to be analyzed after 4 days of incubation. Parameters such as voltage and number of electric pulses, as well as plasmid concentration and developmental stage of the animal were optimized for efficiency. The characteristics of the cultures were assessed by morphology and immunocytochemistry, and cell viability was determined by ethidium homodimer staining. Cell imaging and counting was performed using an automated high-throughput system. This procedure resulted in transfection efficiencies in the order of 22-25% of cultured cells, encompassing both photoreceptors and non-photoreceptor neurons, and without affecting normal cell survival and differentiation. Finally, the feasibility of the technique for cell-autonomous studies of gene function in a biologically relevant context was tested by carrying out gain and loss-of-function experiments for the transcription factor PAX6. Electroporation of a plasmid construct expressing PAX6 resulted in a marked upregulation in the expression levels of this protein that could be measured in the whole culture as well as cell-intrinsically. This was accompanied by a significant decrease in the percentage of cells differentiating as photoreceptors among the transfected population. Conversely, electroporation of an RNAi construct targeting PAX6 resulted in a significant decrease in the levels of this protein, with a concomitant increase in the proportion of photoreceptors. Taken together these results provide strong proof-of-principle of the suitability of this technique for genetic studies in retinal cultures. The combination of the high transfection efficiency obtained by this method with automated high-throughput cell analysis supplies the scientific community with a powerful system for performing functional studies in a cell-autonomous manner.
Wilmer Eye Institute, The Johns Hopkins University School of Medicine, Smith Building 3023, 400 N Broadway, Baltimore, MD 21287-9257, USA. natalia.vergara@jhmi.edu
M Natalia
MN
Vergara
Christian
C
Gutierrez
David R
DR
O'Brien
M Valeria
MV
Canto-Soler
D000818:Animals; D002452:Cell Count; D002642:Chick Embryo; D002645:Chickens; D018274:Electroporation / Q000295:instrumentation* / Q000379:methods*; D005136:Eye Proteins / Q000235:genetics; D018507:Gene Expression Regulation, Developmental / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D018398:Homeodomain Proteins / Q000235:genetics; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics; D010957:Plasmids / Q000235:genetics / Q000493:pharmacokinetics; D061251:Primary Cell Culture / Q000379:methods; D012097:Repressor Proteins / Q000235:genetics; D012160:Retina / Q000166:cytology* / Q000502:physiology*; D014162:Transfection / Q000295:instrumentation* / Q000379:methods*
D005136:Eye Proteins; D018398:Homeodomain Proteins; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D012097:Repressor Proteins; D049452:Green Fluorescent Proteins