L. Maves, March 2001
I. Labeling donors
Inject 1-2-cell stage embryos with fluorescent-Dextran/Biotin dye, ideally into the cytoplasm of 1-cell stage embryos to ensure uptake of dye into all blastomeres. Embryos are injected while in Embryo Medium (EM) (p/s, f/s), and then allowed to continue developing in EM (p/s, f/s).
II. Dechorionation
At about sphere stage, donor and host embryos are transferred into small 1.2% agar dishes in EM (p/s, f/s). Embryos are then manually dechorionated. At this time, some dishes can be moved to room temperature to slow growth, providing a wider window for having embryos at the proper stage for transplantation.
III. Mounting
Place a drop of 2-3% methyl cellulose in Ringeršs (I use 2% for gastrula stages, 3% for post-gastrula) in the center of a transplantation-dedicated glass depression slide. Using a fire-polished pipette, transfer a donor embryo into the methyl cellulose and orient using a clean loop. Transfer a host embryo into the methyl cellulose and orient. Then flood the depression with Ringeršs (p/s, f/s).
IV. Transplanting
Working on a UV stereomicroscope at about 100-150X, focus on the region of interest in the donor embryo. Using a pulled glass needle as a knife, cut out a piece of tissue from the donor (usually about 50-100 cells). I usually make a circular motion with the needle to cut out tissue. If embryos are older (late gastrula), it may be necessary to cut through the skin first-to do this, I use 2 needles to act as a scissors. Once the tissue is freed from the donor, I sweep it, using the needle, to the host embryo. Using the needle as above, tear an opening in the host skin that is roughly the size of the transplant. It is usually not necessary to remove any host tissue. Carefully push the donor piece into the opening in the host. Check briefly with UV light the size and position of the labelled donor tissue.
V. Post-transplantation
Gently submerge the depression slide in EM (p/s, f/s) in a large petri dish. Avoid moving the slides or dishes for at least an hour or two, or at least until embryos are post-gastrulation.
Dextran/Biotin dye: 3% 10k MW fluorescein- or rhodamine-dextran, lysine fixable (Molecular probes, D-1820) and 3% 10k MW Biotin-dextran, lysine fixable (Molecular probes, D-1956) in 0.2M KCl, filtered through a 0.22 um filter.
Embryo Medium (p/s, f/s): to 1 liter of fresh Embryo Medium, add 20 ml of Sigma penicillin (5000 U)/streptomycin (5mg/ml). Filter-sterilize through a Nalgene 75mm filter unit.
Ringeršs (p/s, f/s): Following recipe in The Zebrafish Book:
23.2 ml 5M NaCl final 116 mM NaCl
2.9 ml 1M KCl final 2.9 mM KCl
1.8 ml 1M CaCl2 final 1.8 mM CaCl2
1.19 g Hepes final 5 mM Hepes
bring to about 1L with 2X gd water
pH to 7.2 with 1M NaOH
add 20 ml pen/strep and filter as for EM.
Glass needles: Sutter Instrument Borosilicate with Filament, OD 1.2mm, ID 0.69mm. Pulled using Program 69 on Westerfield puller (makes short, stiff needles).