The pregnant mice were anesthetized either with a combination of isoflurane (1.0% in air) and pentobarbital sodium (80 mg/kg body weight; Somnopentyl, Kyoritsu Seiyaku Corporation, Tokyo, Japan) or with a mixture of medetomidine (37.5 μg/kg; Nippon Zenyaku Kogyo, Fukushima, Japan), midazolam (2 mg/kg; Sandoz, Tokyo, Japan), and butorphanol (0.25 mg/kg; Meiji Seika Pharma, Tokyo, Japan). The uterus was exposed after abdominal incision, and ~2 μl of plasmid was injected into the 4th ventricle, the central canal of the spinal cord, or the cerebral aqueduct of E12.5 or E11.5 embryos. Five square electric pulses (30 V, 50-ms duration at 200-ms intervals) were applied using a forceps-type electrode (CUY650P5 or CUY650P2, Nepa Gene, Japan) connected to a square-pulse generator (CUY21, BEX, Japan).
In ovo EP was performed on HHst16/17 chick embryos essentially as previously described with some modifications (76 (link)). Approximately 0.5 μl of DNA solution at 1 to 2 μg/μl (or in the case of reporter analysis at 5 μg/μl) was injected into the central canal of the spinal cord, and three square electric pulses (25 V, 50-ms duration at 950-ms intervals) were applied using either a parallel platinum electrode (4-mm exposed end and 0.5-mm diameter) connected to a square-pulse generator (CUY21, BEX, Japan) or a 0.5-mm tungsten wire electrode connected to a BTX electroporator (ECM 830).
In ovo EP was performed on HHst16/17 chick embryos essentially as previously described with some modifications (76 (link)). Approximately 0.5 μl of DNA solution at 1 to 2 μg/μl (or in the case of reporter analysis at 5 μg/μl) was injected into the central canal of the spinal cord, and three square electric pulses (25 V, 50-ms duration at 950-ms intervals) were applied using either a parallel platinum electrode (4-mm exposed end and 0.5-mm diameter) connected to a square-pulse generator (CUY21, BEX, Japan) or a 0.5-mm tungsten wire electrode connected to a BTX electroporator (ECM 830).