Pregnant ICR mice were purchased from SLC Japan. Animals were handled in accordance with guidelines established by Keio University, Kyoto University, Tohoku Medical and Pharmaceutical University, FBRI and RIKEN‐BDR. All electroporations in this report were performed on E14 embryos.
In utero electroporation experiments were performed as described previously with minor modifications (Kawauchi et al, 2003 (link)). Pregnant mice were deeply anesthetized and an abdominal or right dorsal incision was made to access the uterus. Approximately 1 μl of plasmid DNA (shRNA experiments: 3 μg/μl, low concentration of Ncad‐sh1023: 1 μg/μl, rescue experiments: 1–10 μg/μl, pCAG‐EGFP: 0.5 μg/μl) in endotoxin‐free TE buffer (Qiagen) containing Fast Green was injected into the lateral ventricle of embryonic brains with a glass micropipette (GD‐1, Narishige). Holding the embryo in utero with forceps‐type electrodes (NEPA GENE or BEX), 50 ms electric pulses of 35 V were delivered five times at intervals of 450 ms with a square electroporator (NEPA21, NEPA GENE or CUY21, BEX). After electroporation, the uterus was placed back into the abdominal cavity, allowing embryos to continue developing. At indicated stages, embryos were harvested and coronal sections of electroporated brains were prepared by using a cryostat (Leica).
In utero electroporation experiments were performed as described previously with minor modifications (Kawauchi et al, 2003 (link)). Pregnant mice were deeply anesthetized and an abdominal or right dorsal incision was made to access the uterus. Approximately 1 μl of plasmid DNA (shRNA experiments: 3 μg/μl, low concentration of Ncad‐sh1023: 1 μg/μl, rescue experiments: 1–10 μg/μl, pCAG‐EGFP: 0.5 μg/μl) in endotoxin‐free TE buffer (Qiagen) containing Fast Green was injected into the lateral ventricle of embryonic brains with a glass micropipette (GD‐1, Narishige). Holding the embryo in utero with forceps‐type electrodes (NEPA GENE or BEX), 50 ms electric pulses of 35 V were delivered five times at intervals of 450 ms with a square electroporator (NEPA21, NEPA GENE or CUY21, BEX). After electroporation, the uterus was placed back into the abdominal cavity, allowing embryos to continue developing. At indicated stages, embryos were harvested and coronal sections of electroporated brains were prepared by using a cryostat (Leica).
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