Injection of Embryos

1. DNA for injection should be the purest available, preferably prepared by Qiagen midi- or maxipreps. DNA is made up in 0.1 M KCl or water at about 25-50 |g/mL final concentration (see Note 1). The DNA solution contains phenol red (0.25%) to give a visual guide regarding how much solution is injected and which embryos have received DNA.

2. Embryos are injected through their chorions at the one-cell stage (see Note 2). Eggs persist at this stage for 10-30 min after fertilization. Cooling to 16°C will delay the first cleavage, providing more time for injection of large collections of embryos.

3. Freshly harvested eggs are rinsed several times in 10% Hank's before transferring to a Petri dish containing 1% agarose made with 10% Hank's and covered with 10% Hank's (see Fig. 1 and Note 3).

4. Align the embryos at the bottom of the ramp in the agarose as shown in Fig. 1C.

5. Injection can be done using a dissecting microscope equipped with transmitted and/or incident light. The injection needle is mounted in a joystick manipulator and connected to a gas pressure injection apparatus (see Note 4).

6. The needles are pulled on a microelectrode puller (see Note 5). The shape of the needle is critical. It should have a sharp tip with a very small aperture, and the shank length should be short so that it is strong enough to penetrate the chorion.

Fig. 1. (A,B) Five microscope slides are stuck together with Superglue and held in a bulldog clip. Two other slides are held together on the top surface of the five slides such that their ends protrude at either side. These are rested on the rim of the bottom part of a 90-mm Petri dish so that the five stuck slides extend a few millimeters into the dish, but do not touch the bottom surface. A 1% agarose solution, made by heating in 10% Hank's, is poured into the dish and allowed to set. The slides are removed, leaving an indentation in the surface of the agarose in the form of a ramp. (C) Zebrafish embryos are added to the dish in 10% Hank's solution with a wide-mouthed, heat-polished glass Pasteur pipet. (D) The embryos are injected by piercing the chorion and rotating each embryo so the needle can be inserted into the single cell above the yolk. If a minimum of Hank's solution is used, the embryos remain in the well when the needle is withdrawn.

Fig. 1. (A,B) Five microscope slides are stuck together with Superglue and held in a bulldog clip. Two other slides are held together on the top surface of the five slides such that their ends protrude at either side. These are rested on the rim of the bottom part of a 90-mm Petri dish so that the five stuck slides extend a few millimeters into the dish, but do not touch the bottom surface. A 1% agarose solution, made by heating in 10% Hank's, is poured into the dish and allowed to set. The slides are removed, leaving an indentation in the surface of the agarose in the form of a ramp. (C) Zebrafish embryos are added to the dish in 10% Hank's solution with a wide-mouthed, heat-polished glass Pasteur pipet. (D) The embryos are injected by piercing the chorion and rotating each embryo so the needle can be inserted into the single cell above the yolk. If a minimum of Hank's solution is used, the embryos remain in the well when the needle is withdrawn.

7. The needles are made from glass capillaries, which contain a glass filament (see Note 6). The filament aids back-filling the needle. The needle is held vertically in a micropipet storage jar with a layer of water in the bottom to keep the chamber humid. A 1-2 pL drop of solution is placed on the blunt end of the capillary, and the solution is slowly drawn down into the capillary along the filament (see Note 7). The tip of the needle is sealed when the capillary is drawn out by the needle puller. The tip must be broken by touching against a fine pair of watchmakers forceps. The tip is best kept immersed in liquid to avoid it becoming blocked.

8. After injection, remove the uninjected or dead embryos, and transfer the remaining embryos to a 35-mm Petri dish with a cushion of 1% agarose covered with 10% Hank's solution containing gentamicin at 20 pg/mL (see Note 8).

9. Incubate the embryos for several hours at 28.5°C (see Note 9). Remove those that have failed to develop or are dead.

10. Some embryos can be examined for transient gene expression 4 h after injection onward.

11. Raise the embryos at low density until they hatch as fry, and then rear to sexual maturity.

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