Methods

1. At least 1 h before collecting the eggs, set up four 35-mm tissue-culture dishes containing 2-3 mL M16 medium and two 35-mm culture dishes containing M16 microdrops (40 | L) covered with liquid paraffin. Allow these to equilibrate in a 37°C incubator gassed with 5% CO2. At the same time, prepare five 35-mm tissue-culture dishes containing 2-3 mL M2 medium and leave at room temperature.

2. Kill the plugged donor females as described in Subheading 5.

3. Lay the animal on its back, and soak the abdomen with 70% ethanol.

4. Pinch up the skin with fingers, cut into the midline, and skin the animal. Cut the body wall, and enter the abdominal tract. Push aside the coils of the gut to reveal one arm of the reproductive tract, which is associated with a fat pad. Identify the coiled oviduct lying between the uterus and the ovary.

5. Gripping the uterus with a watchmaker's forceps, lift up the reproductive tract. Use a pair of fine forceps to puncture the membrane (mesometrium) that joins the reproductive tract to the body wall. Trim the membrane away from the oviduct.

6. Still gripping the uterus, cut between the ovary and the oviduct (cut A in Fig. 3).

7. Transfer the grip to the oviduct, and cut between the uterus and the oviduct (cut B in Fig. 3). The cuts should be made as close to the oviduct as possible.

8. Place the oviduct into one of the dishes of M2 prepared earlier.

9. Dissect the oviduct from the other horn of the reproductive tract, and then proceed with the rest of the female donors. All the oviducts should be placed in the same dish of M2 medium.

10. View the oviducts under the 10-20x magnification of the dissecting stereomicro-scope. The oviduct should appear as mass of opaque coils with a single transparent swollen region, the ampulla (Fig. 4). The ampulla is the target of the egg collection, since, at this stage, it contains the cumulus mass (numerous eggs surrounded by cumulus cells). Eggs may be visible through the walls of the ampulla.

11. Use one pair of sharp watchmaker's forceps to hold the oviduct down, and another to tear the ampulla. The cumulus mass should spill out of the hole. Sometimes, it is necessary to tease the eggs out of the ampulla with forceps. Discard the empty oviduct, and repeat the procedure with the rest of the oviducts.

12. Mix the cumulus mass with 50-100 pL of 10 mg/mL hyaluronidase. Enzymatic digestion is required to separate the eggs from the cumulus cells—a few minutes of treatment is sufficient—the eggs should not be in contact with the enzyme for a longer period. Gently pipeting the cells up and down with a general transfer pipet facilitates the process.

13. Using the general transfer pipet, transfer the eggs to a fresh dish of M2 medium to wash away traces of the hyaluronidase. Repeat in another dish of M2. In each wash, try to leave behind the cumulus cells.

14. Wash the eggs twice in two of the prewarmed dishes of M16 medium and finally, transfer the eggs to the microdrop culture (20-30 eggs/drop).

15. Incubate the eggs at 37°C with 5% CO2 until required for microinjection. It is best to leave the eggs for at least an hour before microinjection.

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