In utero electroporation

Timed pregnant C57Bl/6 or Grin1^flox/flox mice with E12.5 embryos were anesthetized with isoflurane (4.5% induction, 2.5% during the surgery) and uterine horns were successively exposed after a midline laparotomy. Embryos were injected with 200 nL plasmid DNA solution (prepared in 0.9% NaCl, 0.3 mg ml^–1 Fast Green) into the third ventricle through the uterine wall. pCAG-IRES-GFP (1 μg μl^–1, gift from Guillermina López-Bendito) was injected alone or co-electroporated, with a Kcna1 shRNA (2 μg μl^–1). Small hairpin (sh) RNA was purchased from Thermo Scientific (TRC Mouse Kcna1 shRNA, RMM4534-EG16485). pCAG-Cre:GFP was a gift from Connie Cepko³⁹ (Addgene, plasmid 13776) and was injected in Grin1^flox/flox embryos at 2 μg μl^–1. pCAG-KCNA1-IRES-GFP was constructed by inserting the human Kcna1 sequence (Dharmacon, MHS6278-202857646) into the pCAG-IRES-GFP. pCAG-KCNA1-IRES-GFP was injected at 2 μg μl^–1 for Kcna1 overexpression experiments. Embryos were electroporated by holding their head between tweezers-style circular electrodes (3 mm diameter, Sonidel Limited, UK) across the uterus wall, whereas five pulses (35 V, 50 ms duration with 950 ms intervals) were delivered with a square-wave electroporator (Nepa Gene, Sonidel Limited). The uterine horns were returned into the abdominal cavity, the wall and skin were sutured, and the embryos were allowed to continue their normal development until P2 or P7.