Microinjection

1. Fill the micropipet with the DNA or RNA using a microloader (Eppendorf, Germany). The capillary reaction of the inner microfilament should direct the solution to the very tip of the micropipet.

2. Attach the micropipet to the needle holder connected with the micromanipulator (Narishige, Tokyo, Japan). The system is operated using a compressed air cylinder, which delivers pulses controlled by a microinjector (Picospritzer II, General Valve Corp., USA, or any equivalent).

3. Precalibrate the amount injected by counting the number of pulses to expel 1 |L of solution under fixed pressure and duration (e.g., 50 psi and 30 ms). For 200 pulses to deliver 1 |L of solution indicates 5 nL injection volume. Inject about 25 nL DNA or RNA.

4. Use a wide-mouth glass pipet to transfer fertilized embryos (see Notes) into the injection chamber and align them (30-40 embryos) along the trough created -between the slide and the Petri dish. Tilt the injection chamber slightly to collect and remove the excess liquid as much as possible. With a pair of blunt forceps, orient embryosm such that the germinal disk is facing the trough.

5. Injection can be done through the chorion and directly into the cytoplasm. To prevent the germinal disk rotating away from the micropipet tip, it is important to have a relatively steep angle (about 45°) between the pipet and embryo; use the micromanipulator to move the micropipet vertically to penetrate the chorion and the cell membrane. Injection through the yolk and then into the cytoplasm can result in blockage of the micropipet and difficulty in withdrawing the needle from injected embryo.

6. Following injection, tilt the injection chamber, and add embryo medium to the embryos. Transfer the embryos into a clean dish, and incubate them in a humidified incubator at 28.5°C. Later, remove all dead or uncleaved embryos.

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