The overall goal of this procedure is to transplant cells or viruses into mouse fetuses. First, make an incision on the ventral aspect of the mouse. Next, exteriorize the uterus and fetuses, and then perform the in utero injections.
Finally, gently return the uterus back to the peritoneal cavity and close the abdominal fascia and skin. Ultimately, results can be obtained that demonstrate cellular engraftment or gene expression through PCR flow cytometry or immunohistochemistry In uterus stem cell transplantation and gene therapy. Our innovative strategies to treat congenital disorders before birth, we can take advantage of the immature fetal immune system to induce tolerance to allogeneic cells or form proteins.
The mouse model of in utero transplantation allows us to study the biological factors unique to the fetus, which lead to successful engraftment intolerance induction. This technique will now be demonstrated by Amer Nial, a surgeon and postdoctoral fellow in my lab. The preparation of injection pipettes begins with pulling glass pipettes using a pipette puller that has been calibrated for separation of the pipette to occur.
Within 15 seconds, position the pipette in the pipette puller and secure it in place using the top and bottom clamps. Next, press the pull button. The filament will get hot and the pipette will separate.
The pipette will have a taper where it separates cut the end of the pipette, such that the distance from the beginning of the taper to the end of the pipette is 1.04 to 1.05 centimeters. Because the length of the pipette is inversely proportional to the caliber of the pipette orifice. Making a longer pipette will result in a weaker tip that is more susceptible to breakage during the injection and may impact the survival of the injected mouse fetus.
To create a smooth bevel, the pipette tip must be sharpened on a diamond sharpening wheel. Turn the wheel on and then gently lower the pipette on the rotating sharpening wheel. When the pipette is initially cut, it will often break with a natural bevel.
At the tip, periodically reevaluate the pipette through the attached microscope to make sure the pipette is still touching the wheel. Because as the bevel becomes smooth, it will lose contact with the sharpening wheel. Once the bevel is smooth, the pipette tip must be sharpened.
Elevate the pipette such that it is no longer touching the sharpening wheel. Rotate it clockwise by 10 to 50 degrees and place the pipette back on the rotating sharpening wheel for approximately 10 seconds. Be careful not to place the pipette on its edge for too long, or it may develop an uneven surface.
Withdraw the pipette from the sharpening wheel and examine the edge. This step usually needs to be repeated with several small adjustments to create a sharp edge. After completing one side, sharpen the opposite edge in the same manner as was just demonstrated.
This diagram depicts the proper sharpening of the injection pipettes, a bottom view of a properly sharpened needle bevel, and a side view of a pipette that has an uneven edge, a shown here. Finally, use a permanent marker to draw a circumferential line. Every four millimeters starting from where the taper of the pipette begins, these demarcations correspond to five microliters of volume.
After preparing the injection, pipettes and injectable material, you can set up for the injections. First, clean your injection pipette twice with one XPBS, submerse the pipette in PBS and depress the fill button until enough fluid is aspirated to fill the pipette. Then each check the PBS.
Next, prepare your procedure area with a warming blanket, lighting and the necessary surgical instruments. Injections will be done using 2.5 x surgical magnification loops. In this demonstration, although a dissecting microscope can also be used, place the anesthetized mouse on a heating pad in the supine position and affix each limb with tape to secure the animal in place.
After clipping the fur, prep the abdomen with 10%povidone iodine, followed by alcohol, confirmed by a toe pinch that the animal is completely anesthetized. Then make a one centimeter incision in the lower abdomen. The most inferior aspect of the incision should be approximately one centimeter superior to theus incise the skin and the fascia.
Be careful not to injure the intestines and bladder, which are immediately under the thin layer of fascia. Using cotton swabs, gently stretch the fascia and deliver the gr uterus through the incision. Identify the right and left ovaries to make sure you visualize the entire uterus, and then count the total number of fetuses.
Place the uterus back in the abdominal cavity before you proceed so that the fetuses remain warm while you prepare for the injections. Since the injectable materials are generally in a small volume, we usually place them in a small micro fuge tube to be able to fill the needle without breaking the pipette tip. Since the injections are the most difficult aspect of this procedure, a good technique that results in minimal trauma to the uterus is critical to the success of this technique.
The fetuses must be held firmly, yet gently enough to avoid damage. It is also imperative that your movements be steady during injection insertion and withdrawal of the pipette. Draw up the appropriate volume of material for the number of fetuses you plan to inject.
It is important to keep the pipette tips of mers to avoid aspirating your sample into the micro injector tubing. Gently hold a fetus and identify the part of the fetus you plan to inject. In this demonstration, we will inject each fetus in a different body part, the first being the fetal liver.
With your thumb and forefinger, stabilize the fetus within the amniotic cavity so that it does not rotate while you are performing the injection. To inject the liver carefully insert the pipette through the uterus, amnion, and fetal skin, and into the fetal liver. A sharp pipette should pass easily through these tissues.
Inject the desired volume into the fetus and withdraw the pipette gently. It is imperative that your movements be steady during the time of pipette insertion, injection, and withdrawal for intraperitoneal injections. The pipette is also inserted through the uterus, amnion, and fetal skin, but aimed slightly below the fetal liver.
Inject the desired volume for intramuscular injections. Position the fetus to identify the hip and femur and inject the gluteus muscle. For intraventricular injections, it is easy to see the coronal sutures to direct the pipette to the appropriate target for these injections.
Slightly larger caliber pipettes are required to penetrate the skull without breaking the pipette. For studies that involve postnatal harvesting, all fetuses must receive technically perfect injections since there is no way to discern, injected and unin injected fetuses after birth. If injections are done properly, material should not leak out from the amniotic cavity or uterus.
After the fetuses have been injected carefully place the uterus back into the abdominal cavity, making sure it is not twisted on itself or around its vascular supply. Deliver one milliliter of one XPBS into the mother's peritoneal cavity to replace any fluid that was lost during the procedure. Close the incision in two layers with a five oh braided absorbable suture.
First, close the fascia without injuring the underlying bowel or bladder, and then close the skin. Finally, return the injected female to its own individual cage. Place the cage on a warming blanket and poke bedding in the cage.
Place the mouse in a quiet area of the mouse facility where it should not be disturbed until several days after delivery. Minimizing any additional stress for the animal will decrease the chances of preterm labor representative results of donor engraftment after in utero cellular transplantation are shown here.Embryonic. Day 14 fetuses were transplanted with GFP positive hematopoietic stem cells.
Peripheral blood sampling was then performed in a chimeric pup to detect donor cell GFP expression using flow cytometry, which confirmed the presence of donor cells. After watching this video, you should have a good understanding of how to make injection pipettes and perform in utero injections. Once these fundamental techniques are mastered.
This method can be modified to inject fetuses at various gestational ages to study basic developmental and stem cell biology.
