Protocol

Micro In Utero Electroporation for Efficient Gene Targeting in Mouse Embryos

Critical Period Mechanisms Research Group, Institute of Physical and Chemical Research (RIKEN), Brain Science Institute, Saitama 351-0198, Japan

Corresponding author (tshimogori{at}brain.riken.jp)

INTRODUCTION

A mixture of DNA and tracking dye is microinjected into a precisely targeted area of the developing mouse embryo. A pulse generator is used to pass current into the tissue using needle electrodes placed on either side of the injection. Fiberoptic light is used to visualize the procedure. This method can be readily adapted for use on most parts of the embryo. It can be used with many different types of genes, as well as for simultaneously delivering multiple genes.

MATERIALS

Reagents

Acetone

DNA solution (1 µg/µl)

Ethanol, 50%

Fast Green FCF protein staining reagent (Sigma-Aldrich)

Phosphate buffered saline (PBS)

Pregnant mice (embryonic day 10.5 or older)

Sodium pentobarbital (see Anesthetics)

Equipment

Autoclip, 9 mm (ROBOZ)

Cotton gauze

Cotton swabs

Fiberoptic light (Leica)

Forceps (ROBOZ)

Glass capillary tubes (Stoelting Co.)

Metallic pin (WPI 5428)

Micropipette puller

Micro-manipulator (KD scientific)

Nail polish

Platinum wire (A-M Systems)

Plastic tubes

Pulse generator, Model 2100 (A-M systems)

Razor blade

Sandpaper

Scissors (ROBOZ)

Suture, 3-0 DEXON II

Tungsten wire (A-M Systems)

METHOD

Micropipette Preparation

1. Pull glass capillary tubes (Stoelting Co.) using a micropipette puller

2. Pinch the tip with forceps such that the external diameter of the tip is ~20 µm.

Electrode Preparation

3. Encase tungsten and platinum wires in plastic tubes and connect one end to a metallic pin.

4. Sharpen the other tip of each wire with sandpaper.

5. Coat the electrodes with a thin layer of nail polish for insulation.

6. After the nail polish has dried, remove ~200 µm from the tip with acetone-soaked cotton swabs.

Electroporation of mouse embryos in utero

7. Prepare a solution of DNA (1 µg/µl) with Fast Green protein dye according to the manufacturer’s instructions.

8. Anesthetize pregnant mice by intraperitoneal injection of sodium pentobarbital (50 µg/gm body weight).

9. Five to 10 minutes after injection, shave the fur over the abdomen using a razor blade. Wash with 50% ethanol.

10. Make an incision (2.5 cm or less) in the abdominal cavity using fine scissors. Carefully remove all of the uterine horns with forceps. Place them onto PBS-moistened cotton gauze. Place the gauze around the incision.
It is important to keep the uterus moistened with PBS at all times.

11. Wet the tip of the fiberoptic cable and the uterus with warm PBS (37ºC).

12. Hold the fiberoptic light with the index and middle finger, and place the uterus between the fiberoptic light cable and thumb. Squeeze the uterus gently to push the embryos closer to the uterine wall.
(see Troubleshooting)

13. Using a micropipette mounted in a micromanipulator, inject the DNA-Fast Green solution into the target area.
(see Troubleshooting)

14. Insert a fine tungsten (-) electrode and a platinum (+) electrode into the uterus such that the injection site is between the electrodes.

15. Initiate electroporation with three series (three square-wave current pulses each) at 1-second intervals. Each pulse should be 7-10 V for 100 milliseconds.
(see Troubleshooting)

16. Place the uterine horn back into its original location with ~ 500 µl PBS. Close the surgical incision in the uterine wall with suture and close the skin with a 9-mm Autoclip. Keep the animal warm until recovery from anesthesia (~2 hours).
(see Troubleshooting)

Troubleshooting

Problem: The injection site is difficult to visualize.

[Step 12]

Solution: Visualization is achieved by placing the fiberoptic light at a specific angle relative to the embryo. Rotate the embryos into position by rubbing the surface of uterus with a cotton swab soaked in PBS.

Problem: The micropipette damages the uterine wall.

[Step 13]

Solution: The size of the electrodes must be precisely controlled. In order to prevent damage to the uterus, 1 mm from the tip should be no more than 50 µm.

Problem: Electroporation is <50%.

[Step 15]

Solution: Electroporation should be optimized for the age of the embryo and the area of the embryo undergoing electroporation. To maximize transfection efficiency, alter the duration of the pulse, the number of electric pulses, or the pulse voltage as necessary.

Problem: Viability is low.

[Step 16]

Solution: Shorter surgical times give better viability. A maximum surgery time of 30 minutes per pregnant dam is critical.

ACKNOWLEDGMENTS

I thank Dr. N. P. Murphy for critical reading of the manuscript.