Figure 3

In ovo electroporation method targeting E3-E4 chicken retina. Glass capillary tubes were pulled to fabricate needles with a tip opening at 0.1 μm in diameter and a 20 mm taper (A). The needle is loaded with DNA/0.025% fast green solution. Eggs were rotated to release the embryo and the shells were sterilized by wiping with 70% ethanol then windowed using forceps (B). The trajectory of the needle approached the eye from behind the head, toward the beak, and tangent to the retina surface (C). The outermost region of the retina opposite of the main bundle of blood vessels entering the eye (arrowhead, C) was targeted for injection. Successful injection was verified by observing that the subretinal space of the eye was filled with DNA/fast green solution (D). Electroporation was performed with the negative electrode placed above the head of the embryo and deeper in the albumin than the eye. The positive electrode was placed below the spine and on the surface of the albumin (E). Electroporation using this orientation drives the DNA in the subretinal space toward the positive electrode and into the retinal progenitor cells (F). The egg was sealed and incubated until tissue harvest at desired time points. Electroporated retinal tissues were then checked for GFP expression. A wholemount image of a retina with GFP expression at E14 (G) shows axons of ganglion neurons originating in the central retina and extending to the optic nerve (ON). Approximately 1/3 of the central retina was transfected with decreasing levels of GFP expression in more peripheral regions (G, H). Using this method of electroporation at E3-4, the central region of the developing chick retina (area between the two red dotted lines in H) was consistently and stably transfected with pCAG-GFP throughout in ovo developmental stages.