Method of Electroporation for the Early Chick Embryo
Chick embryos have long been one of the favored model systems in the field of embryology and developmental biology. Recent advances in the gene manipulation technologies (Muramatsu et al., 1997; Nakamura et al., 2004) make this model system even more attractive for the developmental biologists (see review by Stern, 2005). Thanks to its two dimensional geometry, easiness in accessibility and observation, and well-established fate maps (e.g. Couly and Le Douarin, 1988; Garcia-Martinez et al., 1993; Hatada and Stern, 1994; Psychoyos and Stern, 1996; Sawada and Aoyama, 1999; Cobos et al., 2001; Lopez-Sanchez et al., 2001; Redkar et al., 2001; Fernandez-Garre et al., 2002; Kimura et al., 2006; Matsushita et al., 2008), it has great advantages especially for studies at the early embryonic stages, such as the processes of gastrulation, neural induction, left-right patterning, etc. For such purposes, a whole embryo culture system, originally invented by Dennis A. T. New (New, 1955), and its derivatives (Flamme, 1987; Sundin and Eichele, 1992; Stern, 1993; Chapman et al., 2001) have been widely used.
Here we describe a method of electroporation for the early chick embryos using the in vitro whole-embryo culture. This method is applicable for some modified version of the New culture, by choosing an appropriate type of electrode. It can be applied for the stage 4 to stage 8 embryos (Hamburger and Hamilton, 1951), and the embryos can be cultured up to stage 17. For a long term study, the tissue of interest may be transplanted to the host embryo in ovo to let it survive for the longer period. This also allows precise positional control of the transgene expression in the host embryo. It should be noted that the younger embryos are the more sensitive to the electric stimuli in general, such that marked deformation of the embryos, even though they are alive, are frequently observed. Therefore, the voltage, pulse duration and numbers, and electrode distance, as well as DNA concentration should be optimized in each actual experimental condition. Cells in either the epiblast/ectoderm from the early stages (stage 4~), or the endoderm at relatively later stages (stage 6~) can be electroporated essentially in a similar way, except for the polarity of the electrodes and administration of the DNA solution. As an example, we previously introduced exogenous genes broadly into the early anterior neural plate to demonstrate that the specific responsiveness for the inductive signals and the regional properties was defined by the homeodomain transcription factors (Kobayashi et al., 2002; Lagutin et al., 2003).
KeywordsChick Embryo Neural Plate Primitive Streak Base Electrode Vitelline Membrane
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