Methods

Preliminary procedure: Loading the oviduct transfer pipet (Fig. 8): Fill the pipet with liquid paraffin oil to the shoulder. The viscosity of the oil affords a greater degree of control over the movement of the eggs. Then, take up a small amount of air, then a small amount of M2, and then another bubble. The eggs are then collected, preferably in a stacked rank with a minimum of medium. A third air bubble is taken up followed by a final column of M2. The total length of the eggs/bubbles/medium should not exceed 2 cm. The loaded pipet can be conveniently stuck on a piece of plasticene on the surgical microscope.

Fig. 8. The tip of an oviduct transfer pipet enlarged to show the arrangement of eggs, air bubbles, and media.

1. Anesthetize a 0.5-d pc pseudopregnant recipient female as in Subheading 2.2.

2. Place the animal ventral side down on the lid of a 9-cm Petri dish if operating on a mouse, but directly onto the stage of the dissecting stereomicroscope if using a rat. Spray the back with 70% ethanol.

3. Comb the hair away from the incision site using a pair of fine forceps. Make a 1-cm transverse cut in the skin about 1 cm left of the spinal cord, at the level of the last rib, using large sharp scissors.

4. For mice, locate the orange-colored ovary beneath the body wall. A 3-5 mm cut should be made through the body wall at a point a few millimeters away from the ovary, using fine scissors. Stretch the cut to prevent bleeding.

5. For rats, the ovary is not normally visible through the body wall. Make a parallel 1-cm cut into the body wall using a pair of fine scissors and stretch to prevent bleeding.

6. Introduce a single stitch into the body wall on one side of the incision, and leave the silk suture in place.

7. Pull out the fat pad joined to the ovary using a pair of fine, blunt forceps. The ovary, uterus, and oviduct will be pulled out as well.

8. Attach an artery clip to the fat pad (avoid the ovary), and position the reproductive tract over the back of the animal such that the coils of the oviduct are exposed and the ovary is toward the left.

9. The mouse should be moved (while on the Petri dish) to the stage of the dissecting stereomicroscope. Illuminate the oviduct with a fiberoptic light source and view under the 10-20x magnification.

10. Orient the oviduct coils to reveal a cavity (Fig. 9) that lies below the ovary and behind the coils of the oviduct. The opening of the oviduct (the infundibulum— target of the transfer process) is located within the cavity behind a transparent membrane—the bursa—which covers the cavity, oviduct, and ovary. Find an area of the membrane, preferably above the infundibulum, that is free of capillaries. Tear it gently with watchmaker's forceps. If the infundibulum cannot be seen through the bursa, just rip the membrane at a convenient point, and continue the search. The infundibulum may be lifted out of the cavity when gently gripped with watchmaker's forceps. Excessive bleeding can be halted with an application of epinephrine (see Subheading 13.4.).

InfundibL usually b within the

Incisio

Arte

InfundibL usually b within the

Incisio

Arte

Ovary

Blood vessels

Oviduct

Bursa

Uterus

Fig. 9. Schematic diagram of the mouse ovary and oviduct prepared for oviduct transfer. The infundibulum is located within the coils of the oviduct and may be accessed by penetrating the transparent membrane that covers the oviduct.

Ovary

Blood vessels

Oviduct

Bursa

Uterus

Fig. 9. Schematic diagram of the mouse ovary and oviduct prepared for oviduct transfer. The infundibulum is located within the coils of the oviduct and may be accessed by penetrating the transparent membrane that covers the oviduct.

11. Prepare the eggs for transfer. Remove a maximum of 20 microinjected eggs from the microdrop culture, and wash them in M2 medium. Load the eggs into an oviduct transfer pipet (Fig. 8) as described in the preliminary procedure.

12. Return to the animal. Use small screws of tissue paper held in the watchmaker's forceps to mop up any excess blood.

13. Grip the tip of the infundibulum with a sharp pair of watchmaker's forceps such that the opening can be accessed by the oviduct transfer pipets. Push the tip of the OTP into the mouth of the infundibulum (the opening is not visible until penetrated and may be located by gentle prodding with the tip of the pipet). Push the pipet into the infundibulum until it has entered the ampulla. The pipet tip must be far enough into the infundibulum so it does not fall out when the eggs are expelled, but not so far in that the opening is against the wall of the ampulla, so restricting the escape of the eggs.

14. Expel the contents of the OTP into the ampulla, and monitor delivery of the eggs by the appearance of the bubbles. When three bubbles have appeared, one can be certain that the eggs have been deposited into the ampulla.

15. Withdraw the pipet. Remove the artery clip, grip the fat pad with a pair of blunt forceps, and return the reproductive tract into the body cavity. Sew up the body wall with one or two stitches, and then clip the skin together with an autoclip.

16. Repeat with the other side of the reproductive tract if the availability of the microinjected eggs allow and the animal is sufficiently anesthesized.

17. If the recipient is a rat, inject it with 50 mg ampicillin (Binotal) intraperitoneally.

This prevents low-grade postsurgery infections and can optimize the number of pups subsequently delivered. This step is not necessary for mice.

18. After confirming that the animal has regained some degree of consciousness and mobility, leave it to recover in a warm, quiet place, and then transfer to an individual cage.

19. Examine the animals regularly during the days after the operation to ensure that they do not pick up any infections. Mouse pups should be delivered 19-20 d after the operation, and rats 21-22 d.

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