title: Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach.
abstract: The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols(1). In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.
authors.affiliation: Wilmer Eye Institute, The Johns Hopkins University School of Medicine.
authors.forename: M Natalia
authors.initials: MN
authors.lastname: Vergara
authors.affiliation: Wilmer Eye Institute, The Johns Hopkins University School of Medicine.
authors.forename: Christian
authors.initials: C
authors.lastname: Gutierrez
authors.affiliation: Wilmer Eye Institute, The Johns Hopkins University School of Medicine; mcantos1@jhmi.edu.
authors.forename: M Valeria
authors.initials: MV
authors.lastname: Canto-Soler
mesh_terms: D000818:Animals; D002642:Chick Embryo; D018274:Electroporation / Q000379:methods*; D020419:Photoreceptor Cells, Vertebrate / Q000166:cytology / Q000502:physiology; D010957:Plasmids / Q000008:administration & dosage / Q000235:genetics; D061251:Primary Cell Culture; D015203:Reproducibility of Results; D012160:Retina / Q000166:cytology / Q000502:physiology*; D014162:Transfection / Q000379:methods*