Transfection into Mouse/Rat Embryos In Utero by Electroporation

Transfection into embryos: brain, spinal cord, inner ear and more.

Transfection into Mouse/Rat Embryos In Utero by Electroporation


Gene transfer into embryonic brains using in utero electroporation technique

1) Materials

    • In Vivo Electroporator: NEPA21/CUY21 SC/CUY21 EDIT (Nepa Gene Co., Ltd.)
    • In Vivo Electrode: CUY650P3/CUY650P5 (Tweezers w/3mm or 5mm diameter platinum disk electrode, Nepa Gene Co., Ltd.)
    • Aspirator tube assembly (Drummond)
    • Optical fiber light (Technolight, Kenko, #KTS-100RSV)
    • Sterile gauze (K-Pine, 7.5cm x 7.5cm)
    • Surgical instruments: Fine forceps x 2, Surgery scissors x 2, Ring forceps, Needle holder, Surgical tape
    • Nylon suture (Nesco, #HT1605NA75)
    • Silk suture (D&G, #112451)
    • Micropipette for DNA injection (make the micropipette by a micropipette puller)

2) In Utero Electroporation Procedure

  1. A 2 cm midline incision is then made in the abdominal wall along the linea alba using a set of forceps and scissors. A piece of sterile gauze with a hole cut in the center is placed over the incision, and one uterine horn is drawn out through the hole in the gauze.
  2. After observing the orientation of the embryos through the wall of the uterine horn, a micropipette is inserted into the lateral ventricle, and 2-5μl of plasmid solution is injected by expiratory pressure using the aspirator tube assembly. When CAG-EGFP is used, a concentration of 1μg/μl is sufficient to visualize the migrating neurons.
  3. After the injection, DNA solution containing 0.01% FastGreen can be seen through the uterine wall (red arrow).
  4. After soaking the uterine horn with PBS, the head of embryo is pinched with a tweezers-type electrode, and electronic pulses are applied with the electroporator.

Electroporation settings for ICR mice

of Pulses

If viability was prioritized over transfection efficiency, the number of pulses can be changed to two.

The actual current is displayed on the screen of the electroporator (NEPA21/CUY21/CUY21E, Nepa Gene). Make sure that the current would become 30-60mA. The current varies according to how the electrode applied or wetness of the uterine horn. Examine the gap between electrodes and the electrode contact areas to fit the current in the appropriate range. If the current is still above the range after the examination, change the voltage setting.

3) GFP expression

in utero electroporation GFP

CAG-EGFP was injected into the both lateral ventricles of E14.5 mouse embryos and electronic pulses (33V, 50msec) were charged four times. 3 days later, the embryos (E17.5) were fixed and the brains were removed and examined under a fluorescence stereomicroscope (Fig. A).
Fluorescence was observed in the lateral region of the hemisphere onto which the anode had been placed and in the medial region of the opposite hemisphere.

Brains were frozen and sliced and the fluorescent image was obtained with a confocal laser microscope (Fig. B). GFP positive cells into which DNA was transferred at the ventricular zone (VZ) migrated to the intermediate zone (IZ) and cortical plate (CP). The arrowheads show the border between VZ and IZ and the border between IZ and CP. Dashed line show the border of tissues.
VZ: Ventricular Zone, IZ: Intermediate Zone, CP: Cortical Plate


Hidenori Tabata and Kazunori Nakajima, Department of Anatomy, Keio University School of Medicine



Drug Delivery and Transfection

Electro Cell Fusion

Fluorescent Staining

Single-Cell/Micro-Particle Transfer

Cell Freezing

Mechanical Vibration