The overall goal of this procedure is the dissection and explant culture of murine allantois for the analysis of allantoic attachment in vitro. This method can help answer key questions in the field of mammalian embryo development, such as the control of allantoic attachment for placenta formation. The main advantage of this technique is that it models the first and critical step in placenta formation in vitro.
Demonstrating the procedure will be Kerstin Hadamek, a Technician from my laboratory. To set up the plates, first add 20 microliters of freshly made Alpha 4/Beta 1 integrin solution into each well. Plan for one allantois per well.
Do not deposit any solution with air bubbles, as this will prevent proper coating. After loading the wells, incubate the plate for an hour in a humidified incubator. After the incubation, aspirate the supernatant from the wells while holding the plate at an incline, so the pipette can gently suck out the solution without touching the well bottoms.
Next, wash the wells twice using 50 microliters of warm culture medium. When removing the washing medium, use gentle suction. Apply 50 microliters of block solution to each well.
Then, incubate the plate for at least 30 minutes until they are needed. Allantois explants are very sticky. So, the block step is critical for the success of the protocol.
After sacrificing a pregnant mouse, disinfect the abdominal area with 70%ethanol. Next, open the abdominal skin by making a small incision at the midline with scissors. Use gloved fingers to hold the skin above and below the incision, and pull the skin apart towards the chest and tail of the mouse.
Now, grasp the peritoneum with forceps and with the other hand, make a V-shaped incision from the anterior towards the posterior. Displace the tissues to access the abdominal cavity, and then push the intestines to the side to access the uterus. Now, locate the two uterine horns.
While holding the cervix with forceps, which is the caudal segment of the uterine horn, use fine scissors to cut the ligaments. Then, detach both uterine horns by severing the oviducts with scissors. Once detached, use forceps to transfer the uterus into a 10 centimeter dish containing room temperature PBS.
In the dish, remove the adipose tissue, vessels, and nerves surrounding the uterine horns using fine forceps and spring scissors. Then, proceed with the fine dissection of the embryos. Perform the embryo dissection under a stereo microscope that has an eight to one zoom range.
Begin with separating the embryos by cutting in between the implantation sites with scissors. Grasp the muscular uterine lining of the uterus buds with forceps and pull it apart to expose the decidua. Next, use forceps to transfer the uterus buds into a new, 10 centimeter dish with PBS.
Now, secure the decidua around the embryo with forceps. The mesometrial pull of the decidua is highly vascularized, and can be wider at its base than the anti-mesometrial region containing the embryo. Now, remove the mesometrial part of the decidua.
Make an incision on the anti-mesometrial side of the decidua with fine forceps, and peel the decidua away with forceps to expose the embryo. Isolate the embryo using a second pair of forceps. Using a micro spoon, collect the embryo.
Deposit the embryo into a drop of PBS in a 3.5 centimeter dish. Then within the dish, transfer into a second drop of PBS. Now, dissect the embryo from the yolk sac using forceps.
Because maternal tissue can contaminate the yolk sac preparation and lead to erroneous genotyping, dissect the parietal yolk sac, including Reichert's membrane and the ectoplacental cone. Now, transfer the isolated yolk sac along with some PBS into a storage tube for molecular analysis. Store these samples at negative 20 degrees Celsius.
The next step in the dissection is to cut the allantois from its basal insertion site at the posterior end of the embryo. This requires the use of fine forceps. If developmental staging is required, count the somite pairs of the embryo under phase-contrast optics.
The chorio-allantoic attachment and fusion occur from E8 to E9, and involves at least six somite pairs. For the allantoides, load the pre-coated plate wells with 40 microliters of DMEM supplemented with fetal bovine serum, L-glutamine, and antibiotics. Then, collect each allantois with about five microliters of PBS using a 200 microliter pipette tip with a wide orifice.
Deposit each into a prepared plate well. Now, incubate the allantoides to the desired time point. Do not move the plate excessively, as the goal is to study their attachment.
At each time point, score their attachment using phase-contrast or differential interference contrast optics. Begin with fully spread explants that have been cultured for 20 to 24 hours. Aspirate the culture medium and add 40 microliters of paraformaldehyde to each well.
After 10 minutes of fixation, aspirate the fixative and wash the tissues three times. For each wash, apply 40 microliters of PBS, wait five minutes, and remove the PBS with gentle suction. Next, permeablize the cells and block non-specific binding sites by adding 40 microliters of PBS, supplemented with detergent and serums.
Let the solution incubate with the tissues for 30 minutes at room temperature. Next, wash out the solution with three applications of PBS. Then, apply 20 microliters of staining solution, containing the anti-CD31 primary antibody.
Conduct the stain overnight at four degrees Celsius. The following morning, wash the tissues three times using PBS. Next, apply 20 microliters of the secondary antibody solution, and incubate the tissues for an hour at room temperature protected from light.
After washing out the antibodies, as before, add 20 microliters of mounting medium to each well. Shield the plate from light and in about one hour, the medium will solidify. Then, save the plates at four degrees Celsius until they can be visualized.
Ex-utero, the distal tip of allantoides isolated from wild type C57 block 6J mice were often attached by six hours. Full attachment of the entire allantois was always observed within 12 hours. There was a diversity of allantois explant and attachment morphologies.
By 24 hours, a CD31-positive vascular plexus was observable in the flattened, circular explants. Thus, this protocol provides a culturing method that also enables the differentiation of allantois explants and at the study of the blood vessel formation in vitro. Once mastered, allantois dissection can be done in less than two hours, if it is performed properly.
The evaluation of allantois explant attachment can be performed within a few minutes on a microscope. Following this procedure, gene trail we did mice, small molecule drugs, therapeutic antibodies. Toxins or pathogens can be screened for.
Potential adverse effects on placenta development.
