The overall goal of this procedure is to transplant extreme anterior domain or EAD facial tissue between xenopus Levi embryos. This is accomplished by first removing the EAD tissue from donor and recipient embryos of different genetic backgrounds. Next, the EAD Explan from the donor embryos are inserted into the faces of the recipient embryos.
In the final step, the transplanted embryos are maintained for 60 to 70 hours post fertilization for observation. Ultimately, brightfield and fluorescence microscopy can be used to observe changes in the c craniofacial development of the transplanted animals. This method can help answer key questions in the C craniofacial development field to improve our understanding of c craniofacial deformities, such as new signals required for oracal development rise from the extreme anterior domain Before the operation load capillary tubing into a needle puller, and then pull four to six needles breaking each needle, such that the two to three millimeter flexible hair-like portion of the glass tip is fully removed.
The tip must be relatively rigid, but narrow enough to be used as a cutting tool. Then light a bunsen burner and place the tip of a glass pasture pipette into the blue part of the flame, rotate the pipette such that the tip melts and the hole completely seals forming a closed rounded end. To make glass bridges use long standard pattern forceps to carefully break off three millimeter by three millimeter chunks of cover slip glass.
Then hold the piece of cover slip with tweezers and place the glass in the flame until all four edges soften and curve downward forming a tiny glass dome. Next line, a plastic 60 millimeter Petri dish with red, white, or yellow modeling clay. And fill the dish with 3%phi.
Call 0.5 XMBS solution. Then use the flamed pasture, your pipe pet tool to make 40 to 60 shallow two to three millimeter depressions about the depth of a stage 20 embryo body into the clay 48 hours prior to the experiment. Obtain eggs from female frogs and fertilize them in vitro.
Then after injecting the embryos with capped membrane, GFP mRNA plus any other message, store them at 15 degrees Celsius for 48 hours until they reach stage 19 to 20. On the day of the transplantation experiment, use a fluorescent microscope to select the injected embryos that show uniform bright fluorescence. After wiping down the operating surface stereo microscope and tools with 70%ethanol, use number 5 45 dumont forceps to remove the Vitale membrane of each selected embryo under a stereo microscope.
Then use a plastic graduated transfer pipette with the tip cut off to move the host embryos into the operating dish. Be careful not to touch the embryos to the air water interface. Insert the embryos posterior side down into the clay depressions and use the forceps to gently close the clay around each embryo base, leaving the head protruding from the depression To remove the donor face first, insert one of the pulled needles into the head of the embryo to the left of the cement gland deeply enough so that it passes from the outside of the embryo through the head and into the fore foregut.
Flick the needle from the left to the right side of the head across the entire width of the cement gland to give a clean cut. Then place the needle at the start of the cut at the left border of the cement gland and flick the needle upward until it reaches the bottom of the left eye to create a vertical cut from the left border of the cement gland to the bottom of the left eye. Next, place the needle at the right border of the cement gland and flick the needle upward until it reaches the bottom of the right eye to create a vertical cut from the right border of the cement gland to the bottom of the right eye.
Then to fully excise the tissue, flick the needle from the bottom of the left eye to the bottom of the right eye, creating a horizontal cut that will free the tissue. Now gently push the excise tissue onto the tip of the needle and lift it through the buffer to the part of the dish containing host embryos. Excise the tissue from the host embryo as just demonstrated discarding the host, EAD explan, and inserting the donor explan into the resulting host whole.
Once the donor tissue is correctly positioned and fully inserted carefully, place one of the previously prepared glass bridges over the embryo's face to hold the transplant in place, inserting the ends of the bridge into the clay to stabilize it. After the operation rest, the embryos undisturbed at room temperature for two to three hours. Then once the transplants have healed carefully remove the glass bridges from the embryos and the clay from around the base of the embryos.
Then use a plastic graduated transfer pipette to gently vacuum the embryos out of their depressions. Transfer the embryos into an appropriately labeled Petri dish, half filled with clean 0.1 XMBS supplemented with Gentamycin and grow the embryos at 15 to 18 degrees Celsius for several days until they reach stage 40. Changing the MBS solution daily view the transplanted tissue by fluorescence to confirm that the explan remains healed in place.
Transplanted tissues should be fully inserted into the host head after transplantation, as just demonstrated and must be correctly sized for the host opening for the transplant to be successful. The EAD tissue should not protrude from the head in any way, as can be observed in these two images of improperly positioned explan. Additionally, the face transplant should not be rotated relative to its position in the donor body as shown here.
After several hours, the transplanted tissue and surrounding face should heal, and by the next day, the embryo should appear as the example shown in these images as just demonstrated fluorescence microscopy can be used to confirm that the transplant remained in place during the healing process. At stage 42, the transplanted control tissue will contribute to the mouth and will remain fluorescent green. After watching this video, you should have a good understanding of how to transplant EAD tissue from one embryo into another.
Facilitating the use of this method for identifying which germ layer or region of the EAD is most crucial to or facial development.
