Oncopeltus fasciatus embryo injection
1. Clear O. fasciatus cages by removing the cotton provided for oviposition and replacing with a new fluffed ball of cotton. Place the new ball of cotton on a piece of paper towel, so it cannot pick up embryos already present in the cage. Allow the bugs to lay for several (3-5) hours.
2. Make agar sheets with 3% food-grade agar. Get STL files to make 3D-printed agar sheet molds here. Try to sustain the boiling of the agar mixture for as long as you can before the mixture boils over, as we have found this helpful in decreasing fungal growth later on.
3. Collect embryos and separate from cotton. Add to Drosophila-style embryo collection basket and add commercial bleach diluted 1:50 in water. Incubate for 3 min, then rinse several times with water. Move the embryos to a wet paper towel in a petri dish.
4. Line up embryos within the grooves of the agar sheets. It helps to keep the embryos fairly wet to make them easier to move. See video below: Lining up embryos for injection. Once the agar sheet is full of embryos, carefully move to a small petri dish, and load another agar sheet. As you go, keep track of the order in which each agar sheet was loaded. Start injection with the first sheet loaded and end with the last sheet loaded. Embryos injected immediately after being placed on the agar sheet may be rather turgid and difficult to inject. After sitting for about 30 min on the agar sheet, they will shrivel slightly and will be much easier to inject.
2. Make agar sheets with 3% food-grade agar. Get STL files to make 3D-printed agar sheet molds here. Try to sustain the boiling of the agar mixture for as long as you can before the mixture boils over, as we have found this helpful in decreasing fungal growth later on.
3. Collect embryos and separate from cotton. Add to Drosophila-style embryo collection basket and add commercial bleach diluted 1:50 in water. Incubate for 3 min, then rinse several times with water. Move the embryos to a wet paper towel in a petri dish.
4. Line up embryos within the grooves of the agar sheets. It helps to keep the embryos fairly wet to make them easier to move. See video below: Lining up embryos for injection. Once the agar sheet is full of embryos, carefully move to a small petri dish, and load another agar sheet. As you go, keep track of the order in which each agar sheet was loaded. Start injection with the first sheet loaded and end with the last sheet loaded. Embryos injected immediately after being placed on the agar sheet may be rather turgid and difficult to inject. After sitting for about 30 min on the agar sheet, they will shrivel slightly and will be much easier to inject.
5. Prepare your injection solution and load into your needle. Insert the needle into your injection apparatus. Break the needle with a pair of fine forceps. Place a sheet of embryos onto an upside down petri dish on the stage of a dissecting microscope. Carefully align the tip of the needle with the uppermost plane of the agar sheet. Using a Narishige IM-400 microinjector, we typically use an injection pressure of ~10 psi, but this always needs to be adjusted for each needle. Insert the needle into the embryo as little as possible (toward the posterior for CRISPR/Cas9 injections, in the center for RNAi injections) and inject just enough fluid to be able to see the injection solution in the embryo when using a dye (we typically use McCormick food coloring diluted ~1:40 in our injection solution). See video below: Injecting Oncopeltus fasciatus embryos
6. When done injecting the entire sheet, move the sheet back to a petri dish, and label with the time, date, and injection solution. Later, count the number of embryos on each sheet so you can calculate hatch rate. When incubating at 25°C, embryos should hatch in about 7-8 days. Fungus growing on the agar surface can be a problem. If this is an issue for you, you can brush the embryos with 70% ethanol to remove the fungus. In our experience, the agar typically dries out during the course of embryogenesis, which reduces fungal growth. You can try lowering the humidity of the incubation chamber or moving the embryos off the agar surface if fungal growth is an issue.