Harvest of One Cell Fertilized Eggs

Collect fertilized one-cell eggs at 14.00 h on the day following mating with stud (see Note 1).

As in the case of culturing fertilized eggs from mice, rat eggs that are in the one- or two-cell stages are maintained in Ml 6 microdrop cultures in a 37°C tissue culture incubator gassed with 5% (v/v) C02. However, when the eggs are manipulated outside the incubator, they are kept in M2 medium.

1. About 1 h before the harvest of the eggs, set up two 35-mm tissue culture dishes containing Ml 6 medium and two 35-mm tissue culture dishes containing small drops of Ml 6 medium covered with liquid paraffin. Allow to equilibrate in the 37°C incubator gassed with 5% (v/v) C02. In addition, prepare four 35-mm tissue culture dishes containing M2 medium and leave at room temperature.

2. Kill female rats (smeared positive for sperm) by cervical dislocation: Hold the animal by its tail and stun it on a hard surface, e g., a table top. Then apply a firm pressure at the neck with a large pair of scissors and pull on the tail. If the animal is too large to be killed in this manner, it should be decapitated using a very large pair of scissors or a rodent guillotine.

3. Lay the rat on its back and swab the abdomen with 70% (v/v) alcohol.

4. Make an incision through the skin and the body wall at the lower half of the abdomen. The coiled oviduct located between the ovary and the uterus can be seen once the intestines are moved to the side (Fig. 1 A).

5 Using a pair of fine forceps, grip the uterus to stretch the reproductive tract and tear the mesometrium (a membrane that joins the reproductive tract to the body wall) with a pair of fine scissors.

6. Make a cut between the ovary and the oviduct and another between the uterus and the oviduct while holding the oviduct with a pair of fine forceps (Fig. IB).

7. Place the oviduct in M2 medium.

8. Remove the oviduct from the other horn of the reproductive tract as described in steps 5-7. Then disssect out the oviducts from the rest of the female rats and place in the same dish of M2 medium.

9. View the oviducts under the 20X magnification of a stereo dissecting microscope. Swollen ampulla with the cumulus cells can be seen under this magnification.

10. Tear the ampulla using two pairs of fine watchmaker's forceps to release the fertilized cells surrounded by cumulus cells from the oviduct. Repeat with the rest of the ampullae.

11. Transfer the cumulus masses to a fresh dish of M2 medium using a general egg transfer pipet. Individual eggs are released from the cumulus cells by incubating the cumulus masses with 100 pL of collagenase solution for a few minutes. Usually, it is necessary to pipet the eggs up and down to remove cumulus cells that stick stubbornly to the eggs.

Fig. 1. Harvest of fertilized one-cell rat eggs.

12. Rinse individual eggs twice in fresh M2 medium to remove traces of enzyme and cell debris.

13. Finally, wash the eggs twice in the M16 medium before transferring to the Ml6 microdrop culture.

14. Incubate the eggs at 37°C in 5% (v/v) C02 prior to microinjection.

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