Bio Protoc 2015 Jun;5(12)
Department of Biology, Miami University and Center for Visual Sciences at Miami University (CVSMU), Oxford, USA.
The chick embryo has prevailed as one of the major models to study developmental biology, cell biology and regeneration. From all the anatomical features of the chick embryo, the eye is one of the most studied. In the chick embryo, the eye develops between 26 and 33 h after incubation (Stages 8-9, Hamburger and Hamilton, 1951). It originates from the posterior region of the forebrain, called the diencephalon. However, the vertebrate eye includes tissues from different origins including surface ectoderm (lens and cornea), anterior neural plate (retina, iris, ciliary body and retinal pigmented epithelium) and neural crest/head mesoderm (stroma of the iris and of the ciliary body as well as choroid, sclera and part of the cornea). After gastrulation, a single eye field originates from the anterior neural plate and is characterized by the expression of eye field transcriptional factors (EFTFs) that orchestrate the program for eye development. Later in development, the eye field separates in two and the optic vesicles form. After several inductive interactions with the lens placode, the optic cup forms. At Stages 14-15, the outer layer of the optic cup becomes the retinal pigmented epithelium (RPE) while the inner layer forms the neuroepithelium that eventually differentiates into the retina. One main advantage of the chick embryo, is the possibility to perform experiments to over-express or to down-regulate gene expression in a place and time specific manner to explore gene function and regulation. The aim of this protocol is to describe the electroporation techniques at Stages 8-12 (anterior neural fold and optic vesicle stages) and Stages 19-26 (eye cup, RPE and neuroepithelium). We provide a full description of the equipment, materials and electrode set up as well as a detailed description of the highly reproducible protocol including some representative results. This protocol has been adapted from our previous publications Luz-Madrigal (2014) and Zhu (2014).