Preparation of tools. TIMING 4hrs, 1d before surgery
a. Surgical Tools (Fig. 3a and b).
CRITICAL STEP Avoid using forceps with sharp tips for surgery. You could easily damage the uterus, causing bleeding and eventually embryos’ death.
b. 1X PBS.
c. Plastic connectors, one for each plasmid that will be used (Fig. 4e)
d. Surgical drapes (mouse: 12cm x 20cm; rat: 15cm x 25cm). Since during autoclaving drapes may melt and stick together, wrap each piece into separate paper towels.
e. Gauzes (4 x 4 cm).
2 Immerge the electrodes in a container with 2% Neutracon overnight for sterilization.
3 Cover the heated surgery platform with bench-guard to avoid burns to the animal.
4 Into two separate boxes, prepare cotton balls soaked with 70% ethanol or betadine.
Preparation of animals for in utero electroporation. TIMING 15min
5 Place the animal in the anesthesia-induction box and set the appropriate level of isoflurane and oxygen to anesthetize the animal (Tab. 1).
6 Place the animal on its back on the heated surgery pad. Set the temperature at 32°C.
! CAUTION Setting temperature above 32°C can cause burns to the animal.
7 Apply the Lacrigel on the eyes of the animal to prevent them from drying during anesthesia.
8 Inject painkiller intramuscularly into one hind paw and the antibiotic into the other.
9 Shave the fur with shaver and vacuum it with the vacuum cleaner.
!CAUTION avoid hurting the nipples of the dam. This will result in poor feeding of the pups.
10 Clean the animal’s skin with cotton balls soaked with either Betadine or ethanol. Make circular movements starting in the center of the abdomen and moving progressively toward the periphery to avoid contamination of the wound. Repeat the procedure 6 times alternating first Betadine and then ethanol.
11 Load the DNA into the needle for injection (Fig. 4a).
CRITICAL STEP Place the tip of the pipette at the very bottom of the needle and load the DNA very slowly to prevent air bubbles. Injection of bubbles will likely results in the embryo’s death.
12 Wear the sterile gloves.
13 Release all the sterile tools from the self-sealing autoclave pouch into the sterile plastic surgery box.
CRITICAL STEP Remember to preserve maximum of sterility. If you have an assistant, ask him/her to unpack tools for you. If you are alone, wear only one sterile glove, open the packages with the free unsterile hand, and release the sterile tools from the pack using the hand with the sterile glove.
In utero electroporation. TIMING 30min
14 Use scissors and cut a hole in the surgical drape with flat shanks-straight and place the drape onto the rodent abdomen. The drape will ensure a sterile environment to the embryos.
15 Cut the skin of the animal using scissors (mouse) or scalpel blade (rat, blade #11). Cut close to the “linea alba”, but not on it, to avoid excessive bleeding.
CRITICAL STEP Do not use scalpel blades for cutting the mouse abdomen. The skin and abdominal wall are very thin. There would be risk of stabbing the embryos and/or internal organs by using the blade.
16 Using shark-tooth tissue forceps, grab the abdominal muscles and make sure with fingers that there are no embryos below the point where you intend to start cutting the abdomen. Then, make a small hole in the abdominal wall using scissors with flat angular shanks and continue cutting until the hole is big enough for and easy exposure of the uterus.
17 Using ring forceps, start to pull out the embryos from the dam abdomen.
CRITICAL STEP Be extremely careful during pulling out of the embryos. With the ring forceps, grab the uterus wall in correspondence of the gap between the yolk sacks of two neighboring embryos. Pull out the first 2/3 embryos with the ring forceps, and then gently use your fingers for pulling out the rest of them. Do it slowly without using force, which can cause damage of the uterus, bleeding, squeezing and eventually death of the embryos.
18 Immediately after uterus exposure and before electroporation, wet embryos with warm sterile PBS to avoid excessive cooling of the embryos and to favor current flow by PBS-saline solution during electroporation (Fig. 5). Repeat the procedure before each electroporation.
19 Inject DNA into one of the brain lateral ventricles using the picopump connected to a foot switch. Keep the needle perpendicular to the head surface. Stop injecting the DNA when the whole ventricle is full and the Fast Green dye is clearly visible through the uterine wall as a half-moon shaped spot (Fig. 4a and b). Electroporation of the cerebellum requires injection in the later ventricle and filling of DNA until reaching the fourth ventricle. For more specificity of transfection, we recommend direct injection into the fourth ventricle (Fig. 4c).
! CAUTION Before injecting the DNA in the embryo’s ventricles, release some DNA from the needle to make sure there are no air bubbles at the tip of the needle. Air bubbles may kill the embryo.
CRITICAL STEP Additional third electrode and injection of both ventricles allow for bilateral transfection during a single electroporation episode (BOX 4).
CRITICAL STEP It is very important to have a sharp needle. If the needle is not perforating the yolk sack easily, replace it with a new one. Never use force to sting the embryo. It will damage their brain. When holding the needle, use sterile gauze to prevent contamination.
CRITICAL STEP Never manipulate embryos excessively. If the injection appears too difficult, leave the embryo as non-injected and move to the next embryo.
20 Electroporate the embryos soon after injection to prevent DNA diffusion to the other ventricle, and decrease of electroporation efficiency. Use appropriate electroporation parameters (Tab.2).
! CAUTION Usage of three electrodes increase the risk of current short circuits, which can kill the embryo. Remember to keep sufficient distance among all electrodes to avoid their physical contact.
CRITICAL STEP The size of the electrodes can significantly influence the efficiency of the electroporation (BOX 1, Fig. 6).
To electroporate different brain structures use the following electrode configurations:
Use the forceps-type electrodes connected by a Y- connector to the positive pole and gently grab both sides of the embryo’s head. Connect the third electrode to the negative pole placed at 0o with respect to the horizontal plane right above bregma (Fig. 7)
B. MOTOR CORTEX
Use the forceps-type electrodes connected by a Y- connector to the negative pole, and gently grab both sides of the embryo’s head. Connect the third electrode to the positive pole, and place as for electroporation of the hippocampus (Fig. 8)
C. PREFRONTAL CORTEX
Use the forceps-types electrode connected by a Y-connector to the negative pole and place it on both sides of the embryo’s head. Connect the third electrode to the positive pole, and place it on the front of the embryo’s head (Fig. 9)
D. VISUAL CORTEX
Use the tweezer type electrode connected by a T- connector to the negative pole and place it on both sides of the embryo’s head. Connect the third electrode to the positive pole, and place it right on (rat) or below (mouse) lambda (Fig. 10)
Use the forceps-type electrodes connected by a T- connector to the negative pole and place it on both sides of the neck at the level of the fourth ventricle. Connect the third electrode to the positive pole, and place it on top of the fourth ventricle (Fig. 11)
CRITICAL STEP To avoid embryo’s death avoid squeezing the uterus and yolk sack containing the embryo during electroporation of all brain areas
21 Use extreme care to handing back the embryos into the dam’s abdominal cavity, and to suture the abdominal wall and skin. For detailed instructions on how to efficiently stitch the abdominal wall and skin, see BOX 5.
CRITICAL STEP Stitches must be tight, otherwise the animal will open its wound.
22 On the stitches, use topic antibiotic cream.
23 After surgery, place the animal belly-down (to favor breathing) into a clean cage with wet food inside the cage (to favor feeding and hydration). Place the cage under the heating lamp (see after-surgery care, BOX 6)