Encyclopedia of Experiments: Biology
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- Place two female and two male fish into a divided breeding tank one night before the injection. The following morning, remove the divider and let the fish breed until you observe eggs at the bottom of the tank. Collect the eggs and wash them with E3 medium containing methylene blue, a fungicide. Observe the eggs under a microscope and remove unfertilized eggs. Unfertilized eggs have a clear yolk membrane, while fertilized eggs have a dark yolk membrane.
Keep 10 embryos in a separate Petri dish as uninjected controls. Using a transfer pipette, align the embryos into the trough of a microinjection plate at room temperature and place it under the microscope. Gently push the microinjection needle through the chorion into the yolk and slowly inject a compound of interest containing a marker such as phenol red. Cytoplasmic flow allows the compound to diffuse into the embryonic cells.
After injection, grow the embryos in E3 medium with methylene blue at 28 degrees Celsius. Keep checking the health of the embryos. Remove any dead or abnormally developing embryos and change the medium daily. In the following protocol, we will use microinjection to deliver a ribonucleoprotein, or RNP complex, into zebrafish embryos.
- First, prepare the zebrafish subjects for breeding the night prior to performing injections as described in the text protocol. Then combine Cas9 protein and sgRNA in a two-to-one ratio. Incubate the solution at room temperature for five minutes while the Cas9 and sgRNA form a ribonucleoprotein complex. Then add 0.5 microliters of 2.5% phenol red solutions in sufficient RNase free water to achieve a final volume of five microliters.
To prepare the injection needle, use a micropipette puller to pull a one-millimeter glass capillary. Then cut the tip of the resulting glass needle with a razor blade to obtain an angled opening. Place the needle in a micromanipulator attached to a microinjector. Using a light microscope, adjust the injection pressure until the needle consistently injects one nanoliter of solution in the Petri dish.
- Before performing embryo microinjection, practice preparing needles and injecting control embryos using a dye-only solution and record the survival after 24 hours of development. You should be able to obtain greater than 90% survival compared to an uninjected control.
- Next, select 10 to 15 embryos as a control population and place them in a separate labeled Petri dish. Using a transfer pipette, carefully line the remaining embryos up on a room-temperature injection plate. Under a dissection microscope, inject one nanoliter of the solution into the yolk sac of each embryo. Then return the injected embryos to a labeled Petri dish and cover them with 1X E3 media with methylene blue. Place the injected embryos in an incubator at 28 degrees Celsius for 24 hours. Then inspect the injected embryos to remove dead or abnormally developing individuals.
