Abstract
One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.
Highlights
One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos
Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps
At midblastula stages the primordial germ cells (PGCs) reside along the margin 7,8, so picking up [50-100] random cells from the margin of a lineage-labeled donor embryo frequently results in the transfer of PGCs
Summary
The transplantation needle should enter the embryo at approximately a 45-degree angle. Avoid taking yolk up into the needle, as it will bind to the mineral oil, which will kill the cells. After the desired number of cells is taken up, reverse the pressure slightly to stop the suction and remove the needle from the embryo. Small adjustments to the position of the host embryo can be made by gently turning it using the transplantation needle, as long as care is taken to not touch the yolk. When expelling the donor cells into the host embryo, avoid introducing a large amount of embryo medium or any mineral oil, as this could interfere with development or kill the embryo. After the cell transfers have been completed, transfer the donor-host pairs to the agar coated wells of a 24 well plate to develop further
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