In Utero Electroporation Based Gene Delivery: A Technique to Inject Plasmid DNA into Embryonic Cerebellar Cells of Murine Embryos

Begin by taking a pregnant anesthetized female mouse model placed in a supine position. Prep the mouse by removing hair from the abdominal region. Incise the skin and underlying muscle layers to expose the uterine horns - the sites that host viable embryos.

Now, pull the uterine horns out of the abdominal cavity. Take a sharp tip glass capillary containing the desired plasmid DNA solution. Locate the brain of the embryo and inject the plasmid DNA solution through the uterus wall into the fourth ventricle. This position is anterior to the cerebellar primordium - the desired location for eventual DNA delivery.

After injection, retract the empty capillary. Next, for electroporation of the DNA injected embryo, position the two electrodes over the uterine wall such that the positive electrode covers the cerebellar primordium and the negative electrode covers the region around the ears.

Apply electric pulses for the desired duration. These pulses make the cerebellar neuronal cells permeable, allowing the plasmid DNA to enter the targeted cells. Finally, place the uterine horns back inside the abdominal cavity at their natural position. Suture the cuts, transfer the mouse to its cage, and allow it to recover.

Begin by loading a borosilicate glass capillary into a micropipette puller and pulling to create a fine tip. Label the capillary tip with black ink for better visualization during the injection and draw a scaling on the glass. Working under a stereomicroscope, use a ruler and sharp needle tweezers to trim the tip to a length of about 6 millimeters and create a sharp, one-sided beveled edge.

Filter a 1% Fast Green stock solution through a 0.22-micron filter to remove dye particles from the solution. Mix the single-guide RNA plasmids in equal parts with the luciferase reporter plasmid to a final concentration of at least 1 microgram per milliliter for each plasmid.

Color the plasma solution with Fast Green at a final concentration of 0.05%. After anesthetizing a pregnant CD-1 mouse with isoflurane, place the mouse on its back on a heating pad and administer analgesic. Apply eye ointment to prevent eye dryness during surgery. Fix the limbs with tape and sterilize the abdomen with a disinfectant.

Cover the mouse with gauze, leaving only the surgical area exposed. After making a skin incision of about 2 centimeters in length, locate the linea alba and use blunt tip tissue scissors to make a slightly smaller second incision through the peritoneum. Moisten the opened abdominal cavity with pre-warmed sterile PBS.

Next, use ring forceps to carefully extract the uterine horns from the abdominal cavity. Grasp the uterine wall at the gap between the yolk sacs of two neighboring embryos and pull gently. Avoid any pressure on the embryo while pulling it through the incision. Once the uterine horns have been extracted, aspirate approximately 10 to 15 microliters of colored plasmid solution with the prepared glass capillary. Avoid any air bubbles during this process.

Gently hold the embryo with ring forceps and locate the neck area. Slowly penetrate the dorsal hindbrain with the tip of the glass capillary and move into the fourth ventricle. Inject about 1 microliter of the plasmid mix. Confirm that the dyed DNA has not leaked from the brain and is visible as a diamond-shaped structure.

Here, it is most important to fill the entire region of the fourth ventricle with plasmid solution to deliver DNA also to the distal part of the cerebellar primordium. At the same time, make sure not to cause any bleeding during that process.

Place platinum tweezers equipped with 5-millimeter diameter electrode plates laterally over the head of the embryo with the negative pole covering the ear and the positive pole positioned at the cerebellar primordium over the uterine wall and apply electric square pulses.

When all embryos have been electroporated, carefully place the uterine horns back into the abdominal cavity and fill with sterile PBS. Let the uterine horn slide into a natural position. Turn off anesthesia and place the animal belly-down into a new cage to recover.