The technique enables studying the function and range of signaling molecules during embryogenesis by transplanting ectopic signaling sources. It also facilitates the efficient generation of germline mutants. The protocol can be applied to blastula stage zebrafish and medaka embryos, and probably also to other embryonic stages and TDO species.
To assemble the transplantation device, connect a lure tip 25 microliter gas tight syringe to a micro pipette holder using the lure lock fitting. Then mount the device directly onto a manual micro manipulator. To use the transplantation device, place the micro manipulator with the assembled device next to a stereo microscope.
Then remove the plunger and insert the transplantation needle. Lower the needle into a dish filled with Ringer's solution at a 45 degree angle until the tip of the needle is immersed. Insert the plunger approximately halfway to flush out the Ringer's solutions, leaving only a small volume of water in the thin tapered part of the needle.
When using a transplantation needle for the first time, coat the inside of the needle by drawing up yoke from a sacrificed embryo and expelling the yoke material completely. Next, using a transplantation needle, gently position the donor embryo, then position the needle opening orthogonal to the embryo surface and carefully pull the plunger to draw the cells into the needle. Once the desired number of cells is drawn in, stop the suction by gently pushing the plunger down and remove the needle from the embryo by jerking the needle to the side in a short and swift motion.
Leave some Ringer's solution on either side of the cell column by restricting the cells to the tapered end of the needle. Clear any remaining yolk or cell debris by slowly moving the plunger up and down and washing the cells with Ringer's solution. Next, move the transplantation dish with the non dominant hand to position the needle orthogonal to the surface of the host embryo.
Apply slight pressure onto the embryo by carefully squeezing it against the walls. Then with a swift sharp movement, pierce the enveloping layer of the host embryo taking care not to scratch the yolk with the needle. Once the needle is inside, gently push the plunger to extrude the column of cells into the embryo, and slowly retract the needle simultaneously.
For transplantation of germ cells, fill a transplantation dish with Ringer's solution, then transfer the dechorionated sphere to dome stage embryos into the transplantation dish and position the host and donor embryos in alternating columns to transplant the cells from one donor embryo to six different host embryos. To carry out the transplantation, orient the embryos with the margin toward the transplantation needle. For germline transplantation, take the source cells from the margin and deposit them into the same location in the host embryo.
Once the transplantation is completed, allow the embryo to recover in Ringer's solution for 30 minutes to one hour. Then using a fluorescence stereo microscope, examine the transplanted cells. Next, transfer the embryos into a 24 well Agarose coated plate with embryo medium grouping all the host embryos that received cells from the same donor into the same well.
Then incubate the embryos at 28 degrees Celsius until the next day. If required, transfer the donor embryos into labeled PCR strips for genotyping. 30 hours post fertilization, screen the host embryos for successful germline transplants under a fluorescence stereo microscope.
The germ cells should be present at the groove above the yolk extension. Grow the successfully transplanted larva to adult hood according to standard husbandry conditions. In successful transplantations, the embryo looks normal and similar in shape and yolk clarity to untransplanted embryos without large tears in the blastoderm.
In contrast, if an embryo is visibly damaged during transplantation, it will not develop normally. Although the transplantation device has mainly been used on zebrafish embryos at blastula stages, the transplantation and cell extirpation can also be carried out in dechorionated blastula stage embryos of the Japanese rice fish medaka. In the specific case of germline transplantation, a good transplantation results in an embryo with a long horizontal column of cells directly above the yolk margin.
However, success of the procedure can only be assessed after 24 to 30 hours of fertilization when the germ cells are distinct in their fluorescence from somatic cells. The primordial germ cells will appear as small fluorescent spheres in the groove directly above the yolk extension. The presence of these germ cells at the correct location indicates successful germline transplantation.
Examples of unsuccessful transplantations are shown here. In the first example, although the germ cells have reached the gonadal mesoderm, as the host embryo is severely deformed, it will not develop normally. In the second example, fluorescent cells are only detected outside the groove.
These cells are either somatic cells or germ cells that failed to migrate correctly. And both cell types will not contribute to the embryo's germ line. It is critical to avoid damage to the transplanted cells and the host embryo.
Cells should be drawn up slowly, be cleared of any remaining yolk, and be inserted carefully. This method provides an easy way to transplant cells in zebrafish embryos, and will be useful for generating ectopic sources and germline mutants. The main advantage of this device, it is low cost, and is a lot and uses.
