The overall goal of this procedure is to transfer pre-implantation embryos into the uterus of a 2.5 DPC pseudo pregnant recipient. This is accomplished by first exposing the ovary, uct, and first section of the uterus. Next, the UCT is punctured with a needle.
Next, a glass capillary pipette is inserted through the hole advancing into the uterine lumen. Finally, the embryos are released into the uterus. Ultimately, the number of pups born is used to show the survival of the pre-implantation embryos transferred.
The main advantage of this technique over a 16 method like uterine embryo transfer is that the uterine wall is not puncture, avoiding bleeding and embryo flow. All animal experiments were approved by the Beltsville area Animal Care and use committees in accordance with the USDA Animal Care and use guidelines. After administering anesthesia and analgesics to a male mouse with proven mating performance and confirming that the animal is properly sedated, place the mouse on a warm stage and use electric clippers to remove the fur from the ventral area between two imaginary transversal lines placed 0.5 centimeters and 2.5 centimeters above the penis.
Use 10%povidone iodine and 70%ethanol to disinfect the shaved area. Next, using dressing serrated forceps to hold the skin in the medial line of the abdomen, perform a 10 to 15 millimeter longitudinal skin incision approximately one centimeter above the penis. Then use dissecting serrated forceps to hold the linear alba and scissors to cut the muscle with micros.
Dissecting serrated forceps grabbed a testicular ed depose pad on one side and pull it to expose the testes vast deens and epididymus. The vast deens is located medial to the testes and it is clearly distinguishable free tube while holding the vasts deens flame dressing forceps until they turn red and use them to cut and cauterize the vasts deens in two points simultaneously, the cut should remove an approximately five millimeter portion and leave two clearly separated Cauterized ends move the testicle, epi, and vast deference back to the abdominal cavity and repeat the procedure on the other testicle. Then with five zero absorbable sutures, make one or two horizontal mattress stitches in the muscle and use one or two wound clippers to suture the skin.
After identifying the male, move it to a cage placed on a warm stage and observe it until it recovers from anesthesia. Record the possible incidences occurring during vasectomy transfer and add antibiotic to the drinking water. To prepare an embryo manipulation, glass pipette, start by polishing the tips of the glass capillaries.
To avoid damaging the pipette holder. Soften a middle portion of the glass capillary by using a fine flame to heat it while using both hands to slightly rotate the capillary. Once the capillary section becomes soft and malleable, withdraw it quickly from the flame and pull both ends to narrow its external diameter to 130 to 150 micrometers After the glass cools down, use a diamond point pencil, abrasive stone or nail file to cut it by lightly scoring the narrow end and pulling from both sides.
The break should be clean and perpendicular. Polish the tip by very quickly flaming leaving a 100 to 130 micrometer aperture pipettes can be stored for later use. After preparing the surgical area and assessing proper anesthesia as described earlier in this video with the animal on a warm stage, use electric clippers to remove the fur from the dorsal area between the knees and the distal ribs.
Move the recipient to a warm stage under the stereo microscope and place it in a prone position lateral to the surgeon. Cover the area with a sterile towel with a hole exposing the shaved area and illuminate the surgical area. Next, while using dressing forceps to hold the skin in a spot located on the cranial, one third of the line between the last rib and the hips and the dorsal one third of the line between the back and the abdomen.
Use scissors to perform a one centimeter transversal incision in the skin. The ovary or the adipose pad surrounding the ovary will be visible through the body wall. Perform a 0.3 to 0.5 centimeter transversal incision in the body wall over the ovary or adipose pad in a spot where the incision does not cut any large blood vessels.
Load the embryo manipulation pipette with cz BH medium, taking a small air bubble. Introducing five to 10 embryos with a minimal amount of medium. And finally, taking up another small air bubble and a small amount of medium.
Use micros dissecting serrated forceps to grab the adipose pad surrounding the ovary and pull it towards the mouse head to expose the ovary, uct, and a small portion of the upper uterus out of the abdominal cavity. Holding the aspirator mouthpiece in the mouth ready to be used with micros dissecting serrated forceps. Move the UCT and expose the utero tubal junction, keeping the utero tubal junction accessible with the non-dominant hand.
Pick up slight curved microdissection forceps and place them just below the utero tubal junction, grabbing the UCT about two millimeters above that portion. Next, holding the utero tubal junction with the slight curved microdissection forceps. Use a 27 gauge needle to puncture the UCT section close to the forceps.
Then insert the embryo manipulation pipette into the orifice and advance through the utero tubal junction to the uterus. Advancing no more than three millimeters into the organ to prevent endometrial damage. Gently blow to release the embryos into the uterus, ensuring that both air bubbles pass through the uterus and without introducing more air.
After removing the pipette, grab the abdominal pad to move the OV duct and ovary back into the abdominal cavity before suturing. The muscle and skin as shown earlier in this video, allow the female to recover similar to the male clips can be removed 10 days after embryo transfer. Refer to the text protocols for further details.
Tails utero tubal embryo transfer provides a means of transferring embryos to the uterus, avoiding some of the complications associated with uterine embryo transfer. In this table, we show results obtained from transferring CD one blast assist subjected to different kinds of manipulations to CD one recipients. Following the protocol described the survival to term or survival to E 15 is similar between embryos.
Simply cultured in vitro from the zygote stage embryos subjected to IPSC injection to generate chimeric pups and embryos that had their zona removed and were exposed to lentivirus seven hours before embryo transfer. Therefore, utero tubal embryo transfer is a reliable technique to transfer particularly complicated embryos such as those lacking the zita and incubated with lentivirus While attempting this procedure, it's important to be as gentle as possible to the recipient and embryos.
