The overall goal of this procedure is to create chimeric embryos by transplanting ectoderm from donor embryos to the developing upper jaw of chick embryos. This is accomplished by first removing the donor derm for transplantation. Then the host site is prepared for engraftment.
The final step of the procedure is to place the donor tissue on the host site and affix the graft in position. Ultimately, results can be obtained that show exclusive engraftment of donor ectoderm, and through molecular and morphological analysis, the function of donor recod derm can be determined. This method can help answer key questions in developmental biology, such as identifying the role of various tissues during morphogenesis.
Demonstrating the procedure will be Dr.Diane, who a senior investigator from our laboratory. Before beginning this procedure, prepare media and ensure all required solutions are accessible and at the correct temperature. Then whilst wearing adequate eye protection, pull micro capillary tubes to a fine point over an alcohol flame.
To make glass pins sharpen tungsten needles by holding them in the flame of a propane torch. Begin extraction of the donor embryo from the shell by placing a piece of tape over the end of the egg. Shell then use the point of a dissection needle to create a small hole in the center of the tape.
Insert a 10 milliliter syringe and an 18 gauge hypodermic needle into the hole, and remove one milliliter of albumin from the egg. Then using a dissection needle, make a small hole on the top of the shell. Place a second piece of tape to cover this hole.
Cut a circular opening in the egg using two pairs of forceps. Grasp the embryo and place it into a dish of ice cold PBS to wash. After the head of the embryo has been removed, place it into a tissue culture dish containing room temperature, serum free DMEM.
Place the dish under the dissecting microscope while holding the head with forceps. Use spring scissors to gross dissect the upper jaw and lagon from the rest of the head. Then using a blade holder with a sharp razor blade, trim the frontal nasal process from the upper jaw.
Use a pipette to transfer the fronton nasal process into a 30 millimeter Petri dish containing a sufficient volume of diss in PBS and incubate on ice for 20 minutes. After the incubation time has elapsed, aspirate the dispa solution without disturbing the tissue. Then completely cover the tissues with DMEM containing 1%BSA.
To stop the digestion, transfer the tissue into a new culture dish. Then working under a dissecting microscope. Hold the tissue with an un sharpened tungsten needle using a sharpened tungsten needle.
Separate the ectoderm by peeling it away from the other tissues. Once prepared, store the graft tissue in DMEM with BSA on ice until the host is ready for transplantation. Using the previously demonstrated method, create a window in the chicken egg.
Place the egg containing the embryo under the dissecting microscope using two pairs of forceps. Grasp the IGN membrane and amnion and gently tear to make a hole and expose the host embryo. Rotate the head by placing a pair of forceps on the right eye and applying pressure while holding the head steady.
Use a tongues and needle to remove the host derm to accommodate the graft. Add two microliters of neutral red to the Petri dish containing the prepared grafts. Use a glass pipette to transfer the graft to the host.
Place the graft in position to replace the removed ectoderm. Insert a glass pin in each corner of the grafted tissue to pin the graft in place Following engraftment. Close the window tightly with tape.
Return the embryo to the incubator until the required stage of development for analysis, this image shows tissue from a quail chick URA that has been stained with anti Q-C-P-N-A quail specific antibody. The grafted tissue is located between the two arrows. There is no evidence of QCPN staining in the meen kind tissue from a mouse chick chime mirror is seen here Again, the grafted tissue is located between the two arrows in C two hybridization with approved specific to the sign B two repetitive sequence.
In mirroring class one genes reveals that expression of sign B two is restricted to the ome comprising the graft shown here as an image of a quail chick chime mirror that has been trichrome stained tri chrome staining renders collagen and bone blue in color. Duplication of the upper jaw skeleton is seen as indicated by the arrow. This image shows a mouse chick chime mirror that has been trichrome stained again.
Duplication of the upper jaw skeleton is seen. This image shows radioactive in C two hybridization of normal BMP seven expression in the mechy of an rafted chick embryo shown here is tissue from a mouse chick chime mirror. It can be seen that BMP seven expression is induced in the mechy adjacent to the graft as indicated by the arrow.
After watching this video, you should have a good understanding of how to manipulate chick embryos to assess mechanisms that regulate development of the front and nasal process.
