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 for Gene Manipulation
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).
Corresponding Organization : Life Science Institute
Other organizations : Tohoku Medical and Pharmaceutical University, Tohoku University
Variable analysis
- Electroporation (performed on E14 embryos)
- Phenotypic outcomes of embryos after electroporation
- Pregnant ICR mice purchased from SLC Japan
- Handling of animals according to guidelines established by Keio University, Kyoto University, Tohoku Medical and Pharmaceutical University, FBRI and RIKEN‐BDR
- Concentration of plasmid DNA injected (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)
- Electric pulse parameters (50 ms, 35 V, 5 times at 450 ms intervals)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
Annotations
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