Artificial Fertilization of Xenopus Eggs

Xenopus females are induced to lay eggs by the injection of 500-1000 IU human chorionic gonadotrophin (HCG) into their dorsal lymph sacs. This tech nique is defined as a "procedure" under the UK Animals (Scientific Procedures) Act of 1986, and requires a Home Office license. Yields of eggs may be improved if the frogs are "primed" a few days before HCG injection with 30 IU of pregnant mare serum gonadotrophin (PMSG). It is prudent to inject two or three frogs in order to be sure of obtaining enough embryos for an experiment. In what follows, it is assumed that the investigator is right-handed.

1. Dissolve 30 IU PMSG in 0.5 mL water and load a 2-mL syringe.

2. Select a female frog (fat ones are usually good). Hold her still on a flat surface. We use the left hand to hold the frog, with the heel of the hand pressed firmly against the bench in front of the frog's head, and with the middle and index fingers between the frog's back legs. If the muscles of the hand are tensed, as if making a "bridge" for a snooker or pool cue, the hand forms a "cage," which restrains the frog without exerting significant pressure on her.

3. Injections are made into the dorsal lymph sac of the animal. Using the right hand, the injection needle should be inserted just beneath the skin on the dorsal surface of the frog's right hindleg. The needle tip should then be moved beneath the row of stitches of the lateral line organs toward the dorsal midline. After injection, the needle tip should be withdrawn slowly along the path of insertion. There should be little or no bleeding, and the frog should not experience any discomfort. It should be returned gently to a marked tank.

4. Two or 3 d after injection of PMSG, frogs should receive injections of 500-1000 IU HCG, using the same technique. They should be kept overnight in the dark, at 18-22°C.

5. To obtain testes, male Xenopus should be killed by heavy anesthesia followed by decapitation and rostral and caudal pithing. The testes are pale, curved structures about 1 cm long, which are positioned on either side of the spine. They may be removed by making an incision in the ventral surface of the animal, and they can be stored in 60% Leibovitz L-15 medium at 4°C for up to 1 wk.

6. Female Xenopus should start to lay eggs the morning after injection. Fresh eggs can be "squeezed" from the frogs by gentle peristalsis of their ventro-lateral surfaces. The eggs should be transferred to a Petri dish and rinsed with distilled water. Then, using a Pasteur pipet, as much liquid as possible should be removed from the eggs.

7. Fertilize the eggs by rubbing them lightly with a piece of dissected testis.

8. Wait 5 min, and flood the eggs with 10% NAM. After about 15 min, the eggs should rotate so that their heavily pigmented animal hemisphere is uppermost. This is a reliable sign of successful fertilization.

9. "Dejelly" the fertilized eggs by incubating them for about 5 min in 2% cysteine hydrochloride. Rinse thoroughly in 10% NAM. Transfer to an agarose-coated Petri dish. Embryos should begin to cleave about 90 min after fertilization. They can be cultured at temperatures between 14 and 23 °C. At the warmer temperature, it takes about 5 h for embryos to reach stage 8 (the midblastula stage), which is when mesoderm induction assays are usually carried out.

3.2. RNA Synthesis

1. Set up transcription reaction at room temperature:

3.2. RNA Synthesis

1. Set up transcription reaction at room temperature:

Linearized DNA (1 mg/mL)

5

|L

10X transcription buffer

5

||L

0.25 M DTT

2.5

|L

10 mM ATP

5

|L

10 mM CTP

5

|L

10 mM UTP

5

|L

1 mM GTP

5

|L

2.5 mM GpppG

10

|L

RNasin

2.5

|L

SP6 RNA polymerase

2.5

|L

RNase-free water

2.5

|L

2. Mix gently, spin briefly to get the components to the bottom of the tube, and incubate at 37°C for 30 min.

3. Add 2.5 |L 10 mM GTP, and incubate for an additional 1 h.

4. Add 5 |L RQ1 DNase I, and incubate at 37°C for 30 min.

5. Extract twice with phenol/chloroform, and ethanol-precipitate twice. Wash the pellet twice with 75% ethanol.

6. Redissolve RNA in 30 |L DEPC-treated water. Measure A280, and adjust RNA concentration to 1 mg/mL.

RNA can be tested for integrity by taking 1 |L of the reaction after step 4 and running it on a 1% RNaase-free TBE gel. There should be a strong RNA band of the appropriate size.

Was this article helpful?

0 0

Post a comment