In-utero Electroporation for Neuronal Transfection

Neuronal transfections were performed by in-utero electroporation of E15 wild-type C57BL/6J mice as described previously [17] (link). Briefly, an E15 pregnant mother was anesthetized using 2% isoflurane and injected with 0.1 mg/kg of buprenorphrine as anesthetic. Embryonic pups within the intact uterus were temporarily removed from the abdomen and injected into the left hemisphere with 1 µl of DNA mixture, containing the appropriate CRISPR-construct and soluble GFP (10∶1), using a ∼50 µm-diameter pipette sharply beveled at 15°–20° (Narishige, Japan), visually confirming the proper site of correct injection by mixing 0.005% fast green with the DNA. To target transfection to the hippocampus, the head of the embryonic pup was placed between paddles of tweezer electrodes (CUY21 electroporator, NEPA GENE, Japan), with the positive terminal covering the lateral surface of the right hemisphere and the negative terminal covering the left hemisphere. Each injected embryo was then subjected to 5×50 ms/35 V electric pulses. Following electroporation, the intact uterus containing the pups was returned to the abdomen, and the mother’s abdomen sutured shut. Recordings were made from transfected pups 14–20 days following birth.