In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock-out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2 . In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4 . Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6 . The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7 , a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.
Video LinkThe video component of this article can be found at https://www.jove.com/video/3024/ Protocol 1. Preparation of capillary and DNA solution for injection 1. Purify plasmid DNA with a Maxi-prep or equivalent method, and make a final concentration of 2-3 μg/μl. Aliquot 100 μg of DNA, add 10μl of fast green dye (1% stock) and TE (10mM Tris Base, 1mM EDTA Solution, pH 8.0) and adjust a volume to 100 μl to make the final DNA concentration for injection mix to 1μg/μl. The concentration of plasmid DNA can be changed depends on the size of plasmid, and also its transfection efficiency, which needs to be tested individually. 2. Spin the DNA solution for 5 minutes at 14,000 RPM at room temperature and collect supernatant to remove any residue. 3. Pull a glass...
