1. Remove 20 fertilized one-cell eggs from microdrop storage in the 37°C, 5% C02 incubator (see Note 4). While observing the eggs using a dissecting microscope, transfer the eggs into M2 medium contained in a 35-mm tissue culture Petri dish. Rinse the eggs in M2. Discharge the eggs into the injection chamber using a mouth-operated general eggtransfer pipet. Observe this procedure under the microscope using the 4X objective. Attempt to keep the eggs grouped together as they are discharged. Locate them in the bottom half of the field of view, just below the holding and injection pipets (Fig. 4). When discharging the eggs into the injection chamber, be sure to avoid the release of air bubbles, which, at best, will obscure the view of the eggs, and, at worst, will result in the loss of the eggs and necessitate the reassembly of the microinjection system.
2 Using the fine vertical micromanipulator controls, readjust the vertical position of the holding and microinjection pipets such that they are located in the same focal plane as the eggs.
3. Using the horizontal joy-stick micromanipulator control, bring the tip of the holding pipet up to an egg. Gently rotate the Agla syringe control such that the egg is drawn onto the tip of the pipet. The egg should be firmly held on the end of the holding pipet, but care should be taken to avoid distorting and, hence, damaging the egg.
4. Move the egg to the center of the field of view, and then switch to the
40X Nomarski objective lens. Focus up and down to locate the two egg pronuclei. The larger of the pronuclei (the male pronucleus) is usually targeted. It may be necessary to move the egg in order to locate the targeted pronucleus in a convenient position for injection. The egg can be turned by gently expelling it from the end of the holding pipet. Then, using the holding pipet, the egg can be rolled into a suitable position before being sucked back onto the pipet tip.
5. Ensure that the egg is tightly held by the holding pipet by applying half a turn to the Agla syringe control. Ensure that the egg is not damaged by the application of too much suction, although distortion of the egg zona pellucida is not a problem.
6. Carefully focus on the targeted pronucleus. Using the right-hand horizontal micromanipulator joy-stick control, bring the microinjection pipet tip up to the zona pellucida. Adjust the fine vertical micromanipulator control to bring the microinjection pipet tip into the same focal plane as the pronucleus. Squeeze hard on the 50-mL injection syringe. It is sometimes possible to see the DNA solution being ejected from the pipet end either by the observation of the mixing with the M2 medium, or by observation of a slight movement of the egg or of contaminating particulate material in the medium. This is a useful indication that the microinjection pipet is not blocked.
7. With one rapid, but smooth movement, introduce the tip of the microinjection needle into the targeted pronucleus by penetrating the zona pellucida and the egg membrane (see Note 5). As soon as the pipet tip appears to be in the nucleus, squeeze hard on the 50-mL injection syringe (operated with the left hand). One of three things will now happen:
a. Successful injection is indicated by the swelling of the pronucleus (Fig. 5). Continue to introduce DNA into the nucleus by exerting pressure on the 50-mL syringe until the nucleus has reached approximately twice its normal volume. Then withdraw the injection pipet in a single, smooth, rapid movement.
b. Failure to penetrate the elastic cell egg membrane is indicated by the appearance at the microinjection pipet tip of a small, clear drop of liquid (Fig. 6). Such is the elasticity of the egg membrane that it can be pushed to form an invagination stretching from one side of the egg to the other. To penetrate the egg membrane, continue to push the microinjection pipet as far as the holding pipet (being careful not to damage the needle by knocking it against the holding pipet tip). Experienced egg microinjectors can "feel" the egg membrane give way. Pull the tip back to the nucleus, and then squeeze on the microinjection syringe again.
Perivitelhne space v Zona pellucida
Perivitelhne space v Zona pellucida
' C? pronucleus ^ pronucleus
Drop of DNA solution invagination
Drop of DNA solution
Fig 6. Failed penetration of the fertilized one-cell egg owing to the elasticity of the egg membrane.
c. A blockage of the microinjection pipet is indicated by the lack of ejection of any material from the pipet tip. The pipet is probably blocked and should be changed. Alternatively, the pipet puller may be producing microinjection pipets with sealed tips or tips with excessively small openings. Try adjusting the pipet puller such that the tip aperture is slightly larger. Finally, it may be necessary to replace the stock of DNA solution if it has become contaminated with particulate material that could block the pipet tip.
Some investigators are able to enlarge the aperture of a blocked or sealed tip by rubbing it gently against the holding pipet. However, the resulting tip may well damage the eggs.
8. Following the withdrawal of the microinjection pipet from the egg, cytoplasmic particles may be observed to flow out of the egg into the peri-vitelline space. This is indicative of egg lysis. If the eggs lyse following two or three successive attempts at injection, the microinjection pipet should be replaced.
9. Switch back to the 4X objective lens, and move the injected egg away from the microinjection pipet. Eject the egg from the holding pipet. Injected eggs should be sorted into two groups—those that have survived injection, and those that have not (Fig. 4).
10. Inject all the eggs in the batch, and then return those that have survived to Ml6 microdrop culture at 37°C in a 5% C02 tissue culture incubator via two washes in 5% C02 equilibrated Ml6 11 Eggs that have survived injection (see Note 6) are returned by oviduct-transfer surgery and returned to the natural environment afforded by a pseudopregnant recipient female (see Chapter 20). Eggs can be transferred into the surrogate recipient on the same day as injection, or they can be transferred following culture overnight when most will have developed to the two-cell stage.
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