In utero electroporation for genetic manipulation in mouse embryos

In utero electroporation was performed as described previously [25 (link)]. Briefly, pregnant mice at E11.8 or E12.5 were deeply anesthetized 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) prior to electroporation. Plasmid DNA was introduced into the central canal of the spinal cord of embryos using a microinjector (IM-31; Narishige, Tokyo, Japan). Round electrodes (CUY650P2, CUY650P0.5; Nepagene, Ichikawa, Japan) were attached to the uterus, and five electric pulses (30–35 V, 50 ms) were applied using an electroporator (CUY21SC; Nepagene). For gene transfer into Brn3a^cKOAP/+ and Brn3a^cKOAP/cKOAP mice, 0.05 mg/mL of pCAG-Cre, together with 0.4 mg/ml of pCAG-nlsEGFP or 0.4 mg/mL of pCAG-nlsEGFP-CAG-Brn3a, were introduced into the neural tube at E11.8. In the case of rescue experiments with Brn3b and Brn3a-POU, 0.05 mg/mL of pCAG-Cre and 0.4 mg/mL of pCAG-nlsEGFP, together with 0.4 mg/mL of pCAG-Brn3b or 0.4 mg/mL of pCAG-FLAG-Brn3a-POU, were introduced. For gene transfer into Brn3a^Cre/+ mice, 0.2 mg/mL of pCAG-LSL-EGFP with or without 0.2 mg/mL of pCAG-Brn3a were introduced into the neural tube at E12.5.