Zebrafish Embryos Microinjection Protocol

   |  June 1, 2023

Zebrafish is a model organism which has been widely used in microinjection experiments. Since it grows faster than any other mammals, it is easier to obtain the embryos. Substances such as DNA and RNA are injected into the developing embryos by microinjection techniques, providing researchers with the fastest and most reliable means to the preparation of genetically modified or mutated zebrafish species.

1. Materials and Equipment for Zebrafish Embryos Microinjection

Materials: Zebrafish embryos, zebrafish embryo culture medium, agarose solution, Petri dish (10cm), chamber, injection sample (e.g., DNA/RNA), capillary glass tube, pipette, etc.

Equipment: Micropipette puller, microinjection pump, micromanipulator, micropipette beveler (if available), microscope, cold light source, incubator.

2. Procedure

2.1 Preparation of the Micropipette Injection Needles

Preparation of suitable injection needles is the first step to the success of zebrafish embryos microinjection. RWD micropipette puller offers customized needles. The typical cone length should be about 8 mm. The micropipette needs to be further sharpened to get a sharper tip to help with the penetration into the embryo. There are two common methods: (1) use a pair of small tweezers to break the tip and get a tip diameter of about 5-10 μm, or (2) place the micropipette in the center of the needle grinder. Then, slowly lower the needle to make contact with the grinding plate at a 45° angle. The tip is ready for injection when it’s beveled.

Zebrafish embryos microinjection needle

2.2 Preparation of the Injection Well

a. Pour 20 ml of the hot 1.5% agarose solution into a 10 cm Petri dish. Wait for it to solidify. 

b. Set the plastic mold (teeth down) into a 20 ml liquid agarose overlay. 

c. After the agarose sets, remove the mold. Add an appropriate amount of medium. Then, cover it with a lid and wrap it in parafilm, and store it at 4°C for later use. 

Preparation of the injection well

2.3 Embryo Preparation

a. The day prior to injection, set up the zebrafish in breeding tanks with dividers in place in a ratio of 1 female to 1 male. The following morning, remove the dividers to allow the zebrafish to mate and lay eggs.

b. Collect the embryos and check their developments under a microscope. Select the embryos at their single-cell stage for injection.

c. Clean the selected embryos with culture medium, then use a pipette to transfer the embryos to the Petri dish with an injection well.

d. Align the embryos with the same cell polarity in the injection well (e.g., animal pole).

e. Remove excess culture medium from the injection well. The amount of medium should be just enough to cover the embryos. This prevents the embryos from floating or sticking to the injection needle during injection and helps the needle to pierce the surface of the chorion. 

Monitoring zebrafish embryo alignment under a microscope

2.4 Embryo Injection

a. Insert the pipette containing the DNA or RNA to be injected into the microinjection pump. Mount the pump onto the micromanipulator, and adjust the needle angle approximately at 45° with respect to the surface of the injection plate. 

b. Adjust the position of the injection needle by using controls on the micromanipulator while observing the embryos and the needle tip under the plane of the field of the microscope for the clearest view. 

c. Move the micromanipulator to force the electrode through the chorion and yolk. Inject an appropriate amount of sample solution into the embryo. When practicing this technique, an appropriate amount of phenol red solution can be added to the injection sample to show the effects after injection. 

d. Withdraw the injection needle from the embryo in a slow and steady manner. Bring the needle tip to the next embryo for injection.

2.5 Embryonic Cells Culture And Observation

a. Transfer the injected embryos to a clean Petri dish. Add fresh culture medium, and incubate them at 28.5°C.

b. Observe the embryo development under a microscope, and pick out the dead embryos (embryos appearing white and flocculent under the microscope) and malformed embryos (as observed). During this incubation period, remove any aborted embryos and replace the culture medium regularly.

3. Tips

a. Preparing a fine and sharp injection needle is crucial for successful zebrafish embryos microinjection. If the opening of the tip is too big, it may cause excessive leakage. On the contrary, a very small opening may impose risks of blockage, undermining the injection efficiency. Neither of these needle conditions can bring the best injection performance. 

b. Excess amount of injection may damage the embryos, while insufficient amount of injection may disrupt the accuracy of quantification and the uniformity of diffusion. The injected volume should be about 10% of the embryo volume, generally around 1 nl.

c. To guarantee an accurate assessment of zebrafish embryo development, the quality and concentration of the injected solution are of paramount importance. Highly concentrated materials could be toxic to the embryos, and impurities found in the injected solution may lead to abnormal embryo development or even death, impacting the final conclusions of the experiment. 

d. The first cleavage of zebrafish embryos occurs approximately 10-30 minutes after fertilization. Microinjection at the one-cell stage should be performed within this time frame. Therefore, it is necessary to prepare for the microinjection procedure in advance, including setting up the injection needles and aligning the embryos.

e. After completing the injection, slowly pull out the injection needle to avoid withdrawing any materials from the fertilized eggs and causing subsequent damage. 

f. After the injection is completed, the breeding of zebrafish should be treated with care. Pay attention to the temperature, water quality, and density of the culture medium in the breeding environment.

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