Preparation of Oocytes

1. Four days (94 to 96 h) before removal of the eggs, induce superovulation of 10 to 15, 3- to 4-week-old female mice by injecting 5 units per female of pregnant mare's serum (PMS) as a stimulating hormone.

2. Inject 5 units per female of human chorionic gonadotrophin (HCG) 75 to 76 h following the injection of PMS. This injection should be made 19 to 20 h prior to harvesting the eggs.

3. After the injection of HCG, cage the females with males overnight by maintaining them on a 12-h light/12-h dark cycle.

4. The next morning, check the females for vaginal plugs. The mated females should have vaginal plugs within 24 h after mating.

5. Kill the superovulated females that have visible, vaginal plugs by cervical dislocation.

6. Clean the body surface with 70% ethanol, dissect the oviducts and transfer them to disposable Petri dishes containing 30 ml of medium A. Carefully transfer the oocytes from the oviducts to the medium.

Note: Under a dissecting microscope, the fertilized eggs can be identified as containing two nuclei that are called pronuclei. The male pronucleus is bigger than that of the female.

7. Aspirate medium A, carefully transfer the oocytes to a fresh Petri dish and incubate them with hyaluronidase for an appropriate time period to remove the cumulus cells.

8. Rinse the oocytes extensively with four changes of medium A and carefully transfer them to a fresh Petri dish containing 10 to 30 ml of medium B. Place the dish in a 37°C incubator with 5% CO2.

Reagents Needed

Medium A

4.78 mM KCl 94.66 mM NaCl 1.19 mM KH2PO4 1.19 mM MgSO4 1.71 mM CaCl2 4.15 mM NaHCO3 23.38 mM Sodium lactate 0.33 mM Sodium pyruvate 5.56 mM Glucose

20.85 mM Hepes, pH 7.4 (adjust with 0.2 N NaOH) BSA, 4 g/l

Penicillin G (potassium salt), 100,000 units/l Streptomycin sulphate, 50 mg/l

Phenol red, 10 mg/l

Dissolve well after each addition. Filter sterilize and store at 4°C. Warm the medium to 37°C prior to use.

Medium B

4.78 mM KCl 94.66 mM NaCl 1.71 mM CaCl2 1.19 mM KH2PO4 25 mM NaHCO3 1.19 mM MgSO4 23.38 mM Sodium lactate 0.33 mM Sodium pyruvate 5.56 mM Glucose BSA, 4 g/l

Penicillin G (potassium salt), 100,000 units/l Streptomycin sulphate, 50 mg/l Phenol red, 10 mg/l

Dissolve well after each addition. Filter-sterilize and store at 4°C. Warm up to 37°C prior to use.

Microinjection of DNA Constructs into Oocytes

The equipment used for microinjection includes: (1) an injection microscope on a vibration-free table (Diaphot TMD microscope with Nomarski optics is available from Nikon Ltd.); (2) an inverted microscope for setting up the injecting chamber, and holding pipette and needle (A SMZ-2B binocular microscope available from Leitz Instruments Co.); (3) two sets of micromanipulators (Leitz Instruments Ltd.); (4) Kopf needle puller model 750 (David Kopf Instruments); (5) a finer needle and a pipette holder; and (6) a Schott-KL-1500 cold light source (Schott).

1. Thoroughly clean a depression slide with teepol-based detergent and extensively rinse it with tap water and distilled water. Rinse it with ethanol and air-dry. Add a drop of culture medium to the depression slide and a drop of liquid paraffin on top of the medium drop.

2. Assemble all parts necessary for the injection according to the manufacturer's instructions. Carry out a pretest of the procedure prior to injection of the DNA sample.

3. Carefully transfer the eggs into the medium drop using a handling pipette that can take up the cells by suction under the microscope.

4. Prepare microinjection needles with Kopf needle puller model 750. Slowly take up the DNA sample into the injection needle by capillary action.

5. Under the microscope, hold the suspended eggs with the holding pipette and use the micromanipulator to insert the needle individually into the male pronucleus, which is larger than the female pronucleus. Slowly inject the DNA sample into the pronucleus. The pressure can be maintained on the DNA sample in the needle by a syringe connected to the needle holder. After swelling of the pronucleus occurs, immediately remove the needle a short distance from the cell. The volume injected is approximately 1 to 2 pl.

6. Transfer the injected eggs to one side and repeat injections for others. About 40 to 60 eggs can be injected in 1 to 2 h, depending on an individual's experience.

7. Transfer the injected eggs into a fresh dish containing medium B and place it in the incubator.

Reimplantation of Injected Eggs into Recipient Female Mice and Generation of Founder Mice

The injected eggs can be reimplanted into recipient female mice right after injection (one-cell stage embryo) or after being incubated in the incubator overnight (two-cell stage embryo). It is recommended that the injected eggs be transferred at the one-cell stage so that the introduced DNA integrates into chromosomes at the same site. Thus, all of the cells in transgenic animals contain the injected DNA. In general, the introduced DNA integrates randomly at a single site. In some cases, however, multiple copies of DNA insertion may occur.

1. Prepare pseudopregnant females by caging four to eight females (6 to 9 weeks old) with vasectomized males (two females per male) on the day they are needed. Thus, the females will be in the correct hormonal stage to allow the introduced embryos to be implanted but none of their own eggs will form embryos. The pseudopregnant females can be distinguished by inspecting the vaginal area for swelling and moistness (see Method A).

2. Carefully make a small incision in the body wall of a half-day pseudopregnant female (e.g., C57B1/CBA F1), and gently pull out the ovary and oviduct from the incision. Hold the oviduct in place with a clip placed on the fat pad attached to the ovary.

3. Under a binocular dissecting microscope, place the injected eggs or embryos into the infundibulum, using a sterile glass transfer pipette. Generally, 10 to 15 injected eggs can be transferred into an oviduct.

4. Carefully place the oviduct and ovary back in place and seal the incision.

5. Recover the foster mothers by briefly warming them under an infrared lamp (do not overheat), and keep two or three in one cage. Live offspring are usually born 18 days after the surgery. These initial offspring are named the founder animals or GO animals in terms of genetics.

6. Allow the offspring to breed in order to maintain the transgenic mice.

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