Method

2.2.1. Preparation of Donor (Quail) Embryo

1. Remove quail eggs from incubator. With the scissors, gently tap near the blunt end of an egg so as to penetrate the shell. Use the tip of the scissors to cut off a small cap of shell near this end, carefully to avoid damaging the yolk.

2. Allow egg white to pour into waste bucket, assisted by the scissors, taking care to avoid damage to the yolk. You may need occasionally to cut through the rather thick albumen using the scissors.

3. Once most of the albumen has been poured off, make sure the embryo is uppermost; if not, turn the yolk by stroking it very gently with the sides of the scissors.

4. Use the scissors to make four cuts into the vitelline membrane around the embryo. If the embryo does not lie exactly in the center of the egg, make the first cut on the side of the embryo nearest the shell, and proceed in this way until all four cuts have been made. Make sure all the cuts meet each other.

5. Pick up the square of embryo/membrane with the spoon/spatula, trying to collect only a minimal amount of yolk.

6. Transfer the yolk/embryo/membrane with the spoon into the large Petri dish with Pannett-Compton saline under a dissecting microscope. With fine forceps, turn the square of yolk/membrane/embryo so that the embryo is uppermost.

7. After the desired number of donor embryos have been placed into the Petri dish, use two pairs of forceps to separate the embryo from adhering yolk. Working at low magnification, pick up a corner of the square of vitelline membrane with one pair of forceps and slowly but steadily fold it back, steadying the yolk with the other pair of forceps. During the whole procedure the membrane and embryo should remain totally submerged in saline. The embryo should be attached to the membrane. If not, peel the membrane completely and then use foceps gently to remove the embryo from the underlying yolk.

8. Pick up the embryo, with or without adhering membrane, with the wide-mouth Pasteur pipet, and transfer it to a 35-mm dish with clean saline for final cleaning and dissection. The edges of the extraembryonic membranes will be perfectly circular, provided that the embryo has not been damaged during the explantation procedure. Put this aside while preparing the host embryo.

2.2.2. Preparation of Host (Chick) Embryo

The following description is for preparing cultures based on the method of New (6) but with some modifications as described in Stern and Ireland (14). The procedure has been adapted from (15,16). You may also follow the method described in Chapter 15 (7). The main difference between these methods and that originally described by New (6) is the use of rings cut from glass tubing, rather than bent from a glass rod with circular cross-section. The advantage of these rings, with rectangular profile, is that they grip the vitelline membrane tightly and therefore allow transfer of the assembly to a flat plastic dish. Above, I have recommended rings of 27 mm outer diameter, because it is easier to wrap the membrane around these for a novice. However, if larger (ca. 30 mm diameter) rings are used, the embryos will develop up to 6-9 h longer. The longevity of the cultured embryo appears to depend both on the amount of thin albumen under the ring and on the length of time for which it can be cultured before the edges of the area opaca reach the ring.

1. Fill the large Pyrex dish about three-fourths full with saline (about 1.5 L).

2. Open an incubated hen's egg by tapping the blunt egg with coarse forceps, and carefully removing pieces of shell. Tip the egg gently to collect the thin albumen in the small beaker (this is required for culturing), and discard the thicker albumen, assisted with the coarse forceps. Try to remove as much albumen as possible, which will simplify the later steps.

3. When yolk is clean and free from adhering albumen, carefully tip it into the saline container, taking care not to damage the vitelline membrane on the edges of the broken shell. The blastoderm should face upwards. If not, carefully turn the yolk with the side of the coarse forceps.

4. Make a cut into the vitelline membrane enveloping the yolk just below the equator. Continue to cut all the way around the circumference of the yolk.

5. With two pairs of fine forceps, slowly but steadily 'peel' the North Pole of the vitelline membrane, all the way off the yolk. Do not stop during this process. The embryo should come off with the membrane. Let the membrane rest on the bottom of the dish, inner face (containing the embryo) pointing upwards.

6. Lower a watch glass and a glass ring into the container. Slide the vitelline membrane, preserving its orientation, onto the watch glass, and arrange the ring over it so that membrane protrudes around the ring. Pull out the assembly from the saline.

7. With fine forceps, work carefully to fold the cut edges of the vitelline membrane over the edge of the ring, all the way around its circumference. Do not pull too tightly but ensure that the bottom of the membrane is smooth and free from wrinkles as you work around the circumference.

8. Place the watch glass over a black surface. Suck off as much fluid as possible from the outside of the ring with the flamed Pasteur pipet. If there is much yolk remaining over and/or around the embryo, wash it carefully with clean saline. Discard any embryos in which the vitelline membrane has been damaged. Leave the host submerged in saline for the operation.

2.2.3. Grafting and Incubation

1. Having prepared both donor quail and host chick embryos, you are ready for the operation. First bring the dish with the donor quail embryo under the microscope and arrange it so that its ventral (endoderm) surface is uppermost. Using two fine needles, carefully cut out the very tip of the primitive streak, cutting through the whole thickness of the embryo but making sure that you do not cut through the vitelline membrane if this is still attached.

2. Lower the magnification of the microscope, keeping track of the excised node, and pick this up with a Gilson P20 fitted with a yellow tip, set to 1-2 ||L.

3. Move the dish with donor embryos away and place the watch glass with the host chick embryo under the microscope. While looking down the microscope, insert the tip of the Gilson under the saline covering the host embryo and gently expel the quail node onto its surface, keeping track of it at all times.

4. Use fine needles to manipulate the donor node to close to the desired grafting site (Fig. 1). Now carefully lift up a portion of the flap of yolky cells (germ wall margin) that covers the inner margin of the area opaca, working outwards from the area pellucida and taking care not to penetrate the ectoderm underneath, which is only one cell thick. This will produce a pocket into which the graft can be inserted.

5. Slide the quail node into the pocket, pushing it as deep as possible so that when the flap of germ wall margin is replaced it will cover the graft completely.

6. Working under the microscope, carefully remove any remaining saline, both inside and outside the ring. During this process, keep watching the graft to make sure that it does not become dislodged. It is important that the embryo and the inside of the ring remain completely dry during incubation.

7. Now pour some thin albumen (about 2-3 mm thick layer) on the bottom of a 35mm plastic dish. Slide the ring with vitelline membrane off the watch glass, and transfer it to the dish, over the pool of egg albumen. Press lightly on the ring with two forceps to allow it to adhere to the dish.

8. If the level of albumen comes close to the edge of the ring, remove the excess. Also aspirate any remaining fluid from inside the ring. It is best if the vitelline membrane bulges upwards, above a good pool of albumen. This will also help to drain off further fluid accumulated during culture to the edges of the ring.

9. Wet the lid of the plastic dish with albumen. Discard the excess, and seal.

10. Place the dish in a plastic box containing a piece of tissue paper or cotton wool wetted in distilled water, seal the box, and place it in an incubator at 38°C.

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