1. Remove about 20 one-celled eggs from storage in M16 medium at 37°C using a general-transfer pipet.
2. Wash the eggs twice in M2 medium. Load them in as small a volume as possible in the transfer pipet, and discharge them into the injection chamber. Observe the entry of the eggs into the chamber using a 4x objective. Try to keep the eggs in a group positioned below the holding and injection pipets. Avoid releasing any air bubbles into the chamber—eggs become obscured or even lost, necessitating the reassembly of the injection chamber.
3. Readjust the vertical positions of the holding and injection pipets so they are in the same plane as the eggs.
4. Bring the tip of the HP close to an egg, and by adjusting the Agla syringe, apply light pressure such that the egg is held onto the tip of the pipet.
5. Bring the egg to the center of the field of vision. Switch to the x40 DIC objective and focus on the egg. Focus up and down to locate the two egg pronuclei. The larger (male) pronucleus is the target and must be positioned for convenient injection. The egg can be moved by gentle expulsion and resuction and rolling it with the holding pipet.
6. Ensure that the egg is tightly held by applying slightly more suction. The zona pellucida can be mildly distorted without harming the egg.
7. Focus on the target pronucleus. Position the microinjection pipet close beneath the egg so that its tip is vertically below the pronucleus. Use the fine vertical micromanipulator control to bring the tip into the same focal plane as the pro-nucleus. Without changing its vertical plane, bring the tip up to the zona pellu-cida at a level horizontal with the pronucleus.
8. Squeeze hard on the 50-mL syringe or if using the automated system, clear the microinjection pipet. It is possible to see DNA solution being ejected by its mixing with the M2 medium, a slight movement of the egg, or the presence of contaminating particulate matter in the medium.
9. Inject the egg. The zone pellucida is easily pierced. When the tip appears to be within the pronucleus, squeeze on the injection syringe. Three things may be observed:
a. The pronucleus swells. This is a successful injection. Apply pressure until the pronucleus is roughly twice its original volume, and then withdraw the pipet in a single smooth rapid movement (Fig. 7).
b. A small clear bubble appears at the tip of the microinjection pipet, and the perivi-telline space may swell. The extremely elastic egg membrane has not been pierced. To penetrate the membrane, continue to push the microinjection pipet as far as the holding pipet, and then pull the tip back into the pronucleus before injecting again. Experienced operators can "feel" the egg membrane give way.
c. Nothing. The injection pipet is probably blocked and should be changed. Alternatively, the pipet puller may be producing microinjection pipet with sealed tips or tips with excessively small openings—adjust the pipet puller. If particulate matter in the DNA stock is suspected, change the DNA stock.
10. Following injection, cytoplasmic granules may flow out into the perivitelline space—indicating egg lysis. If eggs lyse on two or three successive occasions, change the microinjection pipet.
11. Switch back to the 4x objective, and place the injected egg above the holding and microinjection pipet. Injected eggs should be divided into two groups: eggs that have survived injection and those that have not.
12. Eggs should be maintained in M2 medium for a maximum of 15 min. A skilled operator can inject 15-40 eggs in this period. Once all the eggs in a batch have been injected, return the survivors to M16 microdrop culture at 37°C in a 5% CO2 incubator through two washes of M16 equilibrated at 37°C, 5% CO2.
13. Eggs that have survived injection are returned by oviduct transfer to the natural environment afforded by a recipient pseudopregnant female. Eggs can be transferred on the same day as microinjection or transferred following overnight culture.
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