High Speed Extract Preparation

This protocol is an adaptation of Murray (4). Briefly, a crude cytostatic factor (CSF) arrested egg extract (cytoplasm arrested in meiotic metaphase) is prepared. Calcium is then added to allow the extract to progress into interphase, and a high-speed spin is performed to obtain a purer cytoplasmic fraction. Cytochalasin is omitted from the protocol, since carryover of cytochalasin into the final extract used for sperm incubations interferes with normal development of transplant embryos. Use of high-speed rather than crude cytoplas-mic extracts is advantageous, because high-speed extracts promote swelling of added sperm nuclei (and some chromatin decondensation), but do not promote DNA replication. Replication of sperm DNA incubated in these extracts occurs after transplantation of the nucleus into the egg rather than in the extract. Highspeed extract can be stored frozen in small aliquots (at -80°C) and thawed before use.

1. Prime 8-12 female adult X. laevis about 24 h prior to HCG injection by injecting 25-100 U of PMSG into the dorsal lymph sac. Maintain at room temperature. The evening before the extract preparation begins, inject each frog with 500-800 U HCG, and place 2 frogs/container into 2 L 1X MMR. Since one frog with lysing or activating eggs can compromise the whole extract preparation, we prefer to separate the frogs into pairs for the ovulation. The frogs are then placed at 15-18°C overnight (12-14 h). On the next morning, the egg quality from each container is screened before mixing all the eggs and starting the extract preparation. All the eggs released from a frog that lays mottled, lysing, or dying eggs are left out of the extract preparation.

2. All solutions should be prepared before beginning the extract preparation, since the procedure should be carried through all steps promptly once it is initiated; opti mally, the high-speed spin should begin within 45-60 min of dejellying the eggs. Gently expel eggs manually from each frog into a large dish of 1X MMR, and collect unbroken eggs with even pigmentation. Good eggs can also be collected from the 1X MMR in the frog buckets. Total volume of eggs should be 100 mL or greater before dejellying.

3. Remove as much MMR as possible from the eggs. Dejelly eggs in 2% cysteine in XB salts (no HEPES/sucrose). Add a small amount at a time, swirl eggs, and partially replace with fresh cysteine several times during dejellying. Remove broken eggs with a pipet during dejellying. Dejellying can be performed separately for different batches of eggs, and batches that show breakage or egg activation are discarded.

4. Wash eggs in XB (with HEPES/sucrose). We use about 35 mL for each wash, and do four washes.

5. Wash eggs in CSF-XB with protease inhibitors. We do two 25-mL washes.

6. Using a wide-bore Pasteur pipet, transfer eggs into Beckman ultraclear tubes. For these volumes, we typically use 14 x 95 mm tubes (cat. no.: 344060; Beckman, Fullerton, CA 344057). If multiple tubes will be used, try to transfer an equal volume of eggs per tube. Remove as much CSF-XB as possible, and replace with about 1 mL of Versilube F-50.

7. Spin in a clinical centrifuge at room temperature for about 60 s at 1000 rpm (150g) and then 30 s at 2000 rpm (600g). Eggs should be packed after this spin, but unbroken. Versilube should replace the CSF-XB between the eggs, and an inverted meniscus between the Versilube and displaced CSF-XB should be clearly visible. Remove the excess CSF-XB and Versilube, and then balance the tubes.

8. Spin the tubes in rubber adapters for 10 min at 16,000g at 2°C in Sorvall HB-4 or similar swinging bucket rotor to crush the eggs. The eggs should be separated into three layers: lipid (top), cytoplasm (center), and yolk (bottom). Collect the cytoplasmic layer from each tube with an 18-gage needle by inserting the needle at the base of the cytoplasmic layer and withdrawing slowly. Transfer cytoplasm to a fresh Beckman tube on ice. If large volumes of darkly pigmented eggs are used, the cytoplasmic layer may be grayish rather than golden at this step. After a second spin to clarify this extract, it should be golden.

9. Add protease inhibitors to the isolated cytoplasm (do not add cytochalasin); recentrifuge the cytoplasm in Beckman tubes for an additional 10 min at 16,000g to clarify, again using a swinging bucket rotor. Collect the clarified cytoplasm as before. Expect to get obtain 0.75-1 mL cytoplasm/batch of eggs collected from one frog.

10. Add 1/20 vol of the ATP-regenerating system (energy mix). Transfer the clarified cytoplasm into TL100.3 thick-wall polycarbonate tubes (Beckman 349622). Tubes hold about 3 mL each and should be at least half full.

11. Add CaCl2 to each tube to a final concentration of 0.4 mM; this inactivates CSF and pushes the extract into interphase. Incubate at room temperature for 15 min and then balance for the high-speed spin.

12. Spin tubes in a Beckman tabletop TL-100 ultracentrifuge in a TL100.3 rotor (gold top; fixed angle) at 70,000 rpm for 1.5 h at 4°C.

13. The cytoplasm will fractionate into four layers, top to bottom: lipid, cytosol, membranes/mitochondria, and glycogen/ribosomes. Remove the cytosolic layer from each tube (about 1 mL if 2-3 mL were loaded into the tube) by inserting a syringe into the top of the tube through the lipid layer. Transfer this fraction to fresh TL-100 tubes, and spin again at 70,000 for 20 min at 4°C.

14. Aliquot the high-speed cytosol supernatant into 25-|L aliquots in 0.5-mL Eppendorf tubes. Quick-freeze aliquots in liquid nitrogen, and store at -80°C until use. We typically obtain 1-2 mL of high-speed cytosol from preparations of this scale. Sperm nuclei should be incubated in an aliquot of extract and stained with Hoechst as previously described to determine whether extract is effective. If active interphase extract has been prepared, nuclei should swell visibly (thicken and lengthen) within 10 min of addition to extract at room temperature.

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