Tripolar in utero electroporation of the visual cortex was performed as previously 52 (link). Timed-pregnant Long Evans rats were anaesthetized at E17.5 with isoflurane (induction, 3.5%; surgery, 2.5%), and uterine horns were exposed by laparotomy. Expression vectors (pCAG-IRES-EGFP, 1.5 μg/μL in water) and dye Fast Green (0.3 μg/μL; Sigma, St. Louis, MO) were injected (5–6 μL) through the uterine wall into one of the embryos’ lateral ventricles by a 30-G needle. While the embryo’s head was carefully held between standard forceps-type circular electrodes (10 mm diameter; positive poles; Nepa Gene, Chiba, Japan), a third electrode (7×6×1 mm, negative pole) was positioned on the back of the embryo’s head. 5 electrical pulses (amplitude, 50V; duration, 50 ms; intervals, 100 ms) were delivered with a square-wave electroporation generator (CUY21EDIT; Nepa Gene). Uterine horns were returned into the abdominal cavity, and embryos continued their normal development.
Protocol detail
Tripolar In Utero Electroporation of Visual Cortex
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Abdominal cavity
Electrical
Electroporation
Embryos
Fast green
Forceps
Gene
Head
Held
Ires
Isoflurane
Laparotomy
Lateral ventricles
Long evans rats
Needle
Pulses
Surgery
Uterine
Uterine horns
Vectors
Visual cortex
Corresponding Organization :
Other organizations : Italian Institute of Technology, Scuola Normale Superiore, University of Trento
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Variable analysis
independent variables
- Electrical pulses applied to the embryo's head (amplitude: 50V, duration: 50 ms, intervals: 100 ms)
- Not explicitly mentioned
- Timed-pregnant Long Evans rats used
- Isoflurane anesthesia (induction: 3.5%, surgery: 2.5%)
- Injection volume (5-6 μL)
- Injection of expression vectors (pCAG-IRES-EGFP, 1.5 μg/μL) and dye Fast Green (0.3 μg/μL)
- Circular electrodes (10 mm diameter, positive poles) and a third electrode (7×6×1 mm, negative pole) used for electroporation
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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