INTRODUCTIONThe transfection of GFP-expressing constructs into early embryos permits key developmental events such as gastrulation to be dynamically imaged using time-lapse video-microscopy. This protocol describes the ex ovo electroporation of a DNA plasmid into avian embryos as young as stage X, nearly 24 h earlier in development than most electroporation protocols. Compared to in ovo electroporation, the ex ovo method allows easier embryo orientation (the posterior half of the embryo is darker than the anterior half). Thus, positioning of the specimen and consistency of the electroporation site between embryos is improved. Furthermore, nearly all embryos can be electroporated at the same stage using the ex ovo method: If some embryos have not reached a desired stage, it is possible to temporally stop development of those embryos already at the desired stage by keeping them at room temperature while incubating the rest at 37°C until they develop. The method described here uses relatively low voltage, and the electroporation chamber can be made easily, with no specialized equipment required.