This method can help answer key questions in embryology, gene therapy and stem cell transplantation. An advantage to this technique is that it enables analysis of the effects of different experimental therapies on Murine fetuses. Demonstrating this procedure will be doctors Nicholas Ahn, and Barbara Coons, physicians and research fellows in our laboratory.
Before beginning the procedure, drop five to 10 microliters of sterile PBS into and out of the surgical needle tip, two to three times to clear it of any possible debris. Next fill the needle with the reagent of interest at the appropriate experimental volume and concentration. And press the mode button three times on the micro injector to advance to the injection calibration screen.
Add intervals of 10 or 100 milliseconds to adjust the injection time, and press the mode button two more times. Press the balance button and push the foot pedal once. Then press the pedal again and assess what volume of solution is emptied out of the needle per push.
When the micro injector is calibrated to the appropriate experimental volume, fill the needle to the correct level for the injection. When all of the needles have been cleaned, confirm a lack of response to toe pinch of the pregnant two to six month old female mouse. Shave the abdomen, taking care not to damage the nipples, and transfer the animal to a heating pad in the supine position.
Apply ointment to the animal's eyes and secure the limbs to the heating pad with tape. And disinfect the exposed skin. Then inject local anesthetics such as 100 microliters of 0.25%Bupivacaine subcutaneously.
Next, make a one to two centimeter skin incision such that the lower border is no closer than one centimeter to the introitus, and identify the midline of the fascia, taking care not to injure the surrounding epigastric vessels. Using Adson forceps, pinch the fascia without grabbing any of the underlying organs or fetuses, and open the tissue with scissors. Once safely within the abdominal cavity, extend the fascial incision to the length of the skin incision, and use cotton tip applicators to move the intestines into the upper part of the abdomen to expose the gravid uterus.
Extract the uterus through the incision carefully identifying the right and left ovaries to ensure that all of the fetuses are counted. Place the left uterus back into the abdomen so that only the right uterus is exposed, to keep the non injected fetuses warm. And gently grasp at the most lateral amniotic sac between the thumb and index fingers of the non dominant hand.
Position the dissection microscope over the animal and adjust the focus and lighting for a better visualization of the fetus as necessary. For an intravenous injection rotate the uterus so that the vitelline vain, to be injected is parallel to the needle tip. And lay the needle on the uterus at a five degree angle to pierce the uterine wall.
With the tip between the uterine wall and the amniotic sac, place the tip directly atop the vitelline vain and at a nearly tangential angle, glide the needle over the vain until the bevel advances into the vessel. It's imperative that the angle stays low so that the needle does not penetrate the back wall of the vessel. When a spot of blood is observed at the needle tip, to press the foot pedal to inject the full volume of the material of interest into the vessel.
For an intra amniotic injection, rotate the amniotic sac until a location devoid of vessels is found. Point the needle perpendicular to the uterine wall and pierce the needle through the uterus yolk sac, and amniotic sac taking care not penetrate any fetal tissue. Then inject the appropriate volume of material desired.
After either type of injection, withdraw the needle from the injection site once the desired volume is delivered. And proceed to the next fetus until all of the fetuses of the right uterine horn have been injected. Then return the right uterine horn into the abdomen.
And remove the left uterine horn out of the abdomen. When all the fetuses in the left horn have been injected return the entire uterus to the abdomen, taking care to avoid a uterine or intestinal volvulus. Use a disposable transfer pipette to dispense about two milliliters of sterile PBS into the abdomen, to replace any insensible losses.
And use 4-0 polyglactin 910 sutures to close the fascia and abdomen in one continuous layer. Then transfer the mouse to a clean cage under a heat lamp with monitoring until full recumbency. In this training experiment the livers of the fetuses that received in-utero transplantation by a trainee displayed lower fluorescent levels 24 hours after injection, due to a lower engraftment of the transplanted GFP cells, than did livers injected by an experienced instructor.
Flow cytometric analysis for GFP positive donor cells also correlated with the lower level of fluorescents observed under fluorescent microscopy. The ability of viral vectors delivered via the intra-amniotic route to transduce cells in contact with the amniotic fluid. Is exemplified by the transduction of epithelial cells 48 hours following the in utero injection of Adeno virus carrying the GFP transgene in gestational day 12.5 fetuses.
Once mastered this technique can completed in 15-25 minutes per pregnant mouse. After watching this video you should have a good understanding of how to safely and effectively deliver different therapies to mouse fetuses.
