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Products for this protocol

• 1 M CaCl₂
   
  
• XB Salts (20X)
   
  
  • 2 mM CaCl₂
     
    
  • 2 M KCl
     
    
  • 20 mM MgCl₂
     
    
  • Filter-sterilize. Store at 4°C.
     
    
• Protease inhibitor mix (1000X)
   
  
  • Chymostatin
     
    
  • DMSO
     
    
  • Leupeptin
     
    
  • Pepstatin
     
    
  • Dissolve each of the protease inhibitors (chymostatin, leupeptin, and pepstatin) to a final concentration of 10 mg/mL in DMSO. Store the solution in small aliquots at -20°C.
     
    
• CSF-XB buffer with protease inhibitors, freshly prepared and stored on ice
   
  
  • 5 mM EGTA
     
    
  • 10 mM HEPES (pH 7.7)
     
    
  • 1 mM MgCl₂
     
    
  • This is added in addition to the MgCl₂ present in XB salts; the final concentration of MgCl₂ is 2 mM.
     
    
  • 1X Protease inhibitor mix
     
    
  • 50 mM sucrose
     
    
  • 1X XB salts
     
    
• Cysteine solution (1X XB salts containing 2.0% L-cysteine hydrochloride 1-hydrate, pH 7.8), freshly prepared
   
  
• Energy mix
   
  
  • 20 mM ATP
     
    
  • 150 mM creatine phosphate
     
    
  • 20 mM MgCl₂
     
    
  • Store in 0.1-ml aliquots at -20°C.
     
    
• Extract buffer, freshly prepared and stored on ice
   
  
  • 10 mM HEPES (pH 7.7)
     
    
  • 50 mM sucrose
     
    
  • 1X XB salts
     
    
• 10 mg/mL Hoechst No. 33342, diluted 1:100 prior to use (optional; see Step 21)
   
  
• 1000 U/mL human chorionic gonadotropin (HCG)
   
  
• Liquid nitrogen
   
  
• 10X Marc's modified Ringer's (MMR), diluted to 1X prior to use
   
  
  • 20 mM CaCl₂
     
    
  • 50 mM HEPES (pH 7.5)
     
    
  • 20 mM KCl
     
    
  • 10 mM MgCl₂
     
    
  • 1 M NaCl
     
    
  • Adjust pH with NaOH to 7.5. Sterilize by autoclaving.
     
    
• 100 U/mL pregnant mare serum gonadotropin (PMSG)
   
  
• Xenopus laevis, adult female
   
  

• Beakers for egg collection (see Step 5)
   
  
• Buckets or containers for holding female frogs (e.g., 4-L plastic beakers with mesh lids)
   
  
• Needles (18 and 26 gauge)
   
  
• Pasteur pipette, wide bore
   
  
• Syringes (1 mL)
   
  
• Tubes, microcentrifuge (0.5 mL)
   
  
• Tubes, thick-wall polycarbonate (Beckman, 349622)
   
  
• Tubes, ultraclear (14 x 95 mm; Beckman, 344060)
   
  
• Ultracentrifuge and rotors (e.g., Beckman TL-100 with rotors SW 40 Ti and TLA-100.3)
   
  

1. About 3-5 d prior to HCG injection, prime 8-12 adult female X. laevis by injecting 50 U of PMSG into the dorsal lymph sac using a 26-gauge needle.
    
   
2. The evening before extract preparation, inject each frog with 500 U HCG.
    
   
3. Place the frogs into 2 L of 1X MMR (2 frogs/container), and keep them at 15°C-18°C overnight (12-14 h).
    
   Because one frog with lysing or activating eggs can compromise the whole extract preparation, we prefer to separate the frogs into pairs for the ovulation.
    
   
4. The next morning, screen the quality of the eggs from each container before mixing all the eggs. Leave out all the eggs released in any container with signs of mottled, lysing, or dying eggs from the extract preparation, as these can affect the quality of the extracts obtained.
    
   
5. Gently, manually expel eggs from each frog into large beakers containing 1X MMR.
    
   
6. Collect unbroken eggs with even pigmentation. Good eggs can also be collected from the 1X MMR in the frog buckets.
    
   The total volume of eggs should be >100 mL from the 8-12 females.
    
   
7. To dejelly the eggs, remove as much MMR as possible from the eggs. Add a small amount of cysteine solution, and swirl the eggs. Replace with fresh cysteine solution several times during dejellying.
    
   Dejellying should be initiated separately for different batches of eggs. Discard any batches that show breakage or premature egg activation. Egg activation can be recognized by constriction of the animal pole region of the eggs. The rest of the eggs can then be combined.
    
   
8. Wash the eggs in ~35 mL of extract buffer, four times.
    
   
9. Wash the eggs in 25 mL of CSF-XB with protease inhibitors, twice.
    
   
10. Using a wide-bore Pasteur pipette, transfer the eggs into Beckman ultraclear tubes. Allow the eggs to settle. Remove as much CSF-XB as possible.
     
    
11. Centrifuge the eggs using a Beckman SW 40 Ti rotor at 1000 rpm (150g) for ~60 sec at 4°C. Remove excess solution from the top of the packed eggs. This step packs the eggs, but does not crush them yet.
     
    
High-Speed Extraction of Xenopus Eggs
 

12. Centrifuge the eggs at 10,000 rpm (16,000g) for 10 min at 4°C to crush the eggs. The eggs should be separated into three layers: lipid (top), cytoplasm (center), and yolk (bottom).
     
    
13. Collect the cytoplasmic layer from each tube with an 18-gauge needle by inserting the needle at the base of the cytoplasmic layer. Transfer the cytoplasm to a fresh ultraclear Beckman tube on ice.
     
    
14. Add protease inhibitors to the isolated cytoplasm to a final concentration of 1X. Recentrifuge the cytoplasm at 16,000g for 10 min at 4°C.
     
    
15. Collect the clarified cytoplasm as described in Step 13.
     
    Expect to obtain 0.75-1 mL cytoplasm/frog.
     
    
16. Add 1/20 of the extract volume of energy mix to the sample. Transfer the cytoplasm into thick-walled polycarbonate tubes.
     
    Tubes hold about 3 mL each and should be at least half full.
     
    
17. Add 1 M CaCl₂ to each tube to a final concentration of 0.4 mM. Incubate the tubes for 15 min at room temperature.
     
    This inactivates CSF and pushes the extract into interphase.
     
    
18. Balance the tubes. Centrifuge them in a Beckman TL-100 ultracentrifuge using a TLA-100.3 rotor at 70,000 rpm for 1.5 h at 4°C.
     
    The cytoplasm will fractionate into four layers, top to bottom: lipid, cytosol, membrane/mitochondria, and glycogen/ribosomes.
     
    
19. Remove the cytosolic layer from each tube (~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.
     
    
20. Transfer the cytosolic fraction to fresh tubes. Recentrifuge the samples at 70,000 rpm for 20 min at 4°C.
     
    
21. Aliquot the supernatant into 25-µL aliquots in 0.5-mL tubes. Quick-freeze the aliquots in liquid nitrogen. Store them at -80°C until use.
     
    To test the quality of the extracts, add sperm nuclei to an aliquot of extract and stain with Hoechst to determine whether the nuclei visibly swell (i.e., thicken and lengthen) within 10 min of addition at room temperature.
     
    

• 10% bovine serum albumin (BSA; Sigma, A7906), freshly prepared and stored on ice
   
  
• 10 mg/mL Hoechst No. 33342, diluted 1:00 prior to use
   
  
• 1000 U/mL human chorionic gonadotropin (HCG)
   
  
• Liquid nitrogen
   
  
• 10 mg/mL L-{alpha}-lysolecithin, egg yolk (Calbiochem, 440154)
   
  Store the solid stock at -20°C. Discard the stock powder if it becomes sticky. Prepare the 10 mg/mL solution immediately before use at room temperature.
   
  
• 10X Marc's modified Ringer's (MMR), diluted to 1X prior to use and stored on ice
   
  
  • 20 mM CaCl₂
     
    
  • 50 mM HEPES (pH 7.5)
     
    
  • 20 mM KCl
     
    
  • 10 mM MgCl₂
     
    
  • 1 M NaCl Adjust pH with NaOH to 7.5. Sterilize by autoclaving.
     
    
• 2X nuclear preparation buffer (NPB), freshly prepared (to a concentration of 1X) and stored on ice
  • 2 mM dithiothreitol
     
    
  • 2 mM EDTA (pH 8.0)
     
    
  • 30 mM HEPES
     
    
  • 1 mM spermidine trihydrochloride
     
    
  • 0.4 mM spermine tetrahydrochloride
     
    
  • 500 mM sucrose
     
    
• 100 U/mL pregnant mare serum gonadotropin (PMSG)
   
  
• Sperm dilution buffer (SDB)
   
  
  • 75 mM KCl
     
    
  • 0.5 mM spermidine trihydrochloride
     
    
  • 0.2 mM spermine tetrahydrochloride
     
    
  • 250 mM sucrose
     
    
  • Add ~80 µL of 0.1 N NaOH per 20 mL of solution to titrate the pH to 7.3-7.5. Store 0.5-1 mL aliquots at -20°C.
     
    
• Sperm storage buffer (SSB), freshly prepared and stored on ice
   
  
  • 0.3% bovine serum albumin (BSA)
     
    
  • 30% glycerol
     
    
  • 1X nuclear preparation buffer (NPB)
     
    
• 0.1% tricaine methanesulfonate, prepared in 0.1% sodium bicarbonate
   
  
• Xenopus laevis, adult males
   
  

• Centrifuge and rotor (e.g., Sorvall HB-4 swinging bucket rotor)
   
  
• Cheesecloth
   
  
• Dissection tools: forceps and scissors
   
  
• Fluorescence microscope
   
  
• Funnel
   
  
• Gloves, latex
   
  
• Hemocytometer
   
  
• Needles (26 gauge)
   
  
• Paper towels
   
  
• Petri dishes (60 mm)
   
  
• Pipettes, plastic (5 and 10 mL)
   
  
• Pipetman tips (1 mL and 200 µL)
   
  
• Razor blade
   
  
• Syringes (1 mL)
   
  
• Tubes (14 mL; Falcon, 2059)
   
  
• Tubes, microcentrifuge (1.5 mL)
   
  

1. About 3-5 d prior to HCG injection, prime one or two male X. laevis by injection with 50 U of PMSG using a 26-gauge needle fitted to a 1-mL syringe.
    
   
2. Twelve to fifteen hours before nuclei preparation, inject the males with 500 U of HCG.
    
   
3. Anesthetize the male by immersion in tricaine for at least 20 min. Pith it.
    
   
4. Isolate the testes with dissecting scissors. Roll them on a paper towel to remove blood, blood vessels, and adhering fat.
    
   For details on this procedure, please consult Isolating Xenopus laevis Testes.
    
   
5. Wash the testes briefly in a 60-mm Petri dish containing cold 1X MMR. Remove any attached pieces of fat or debris with forceps.
    
   
6. Transfer the cleaned testes to a dry 60-mm Petri dish. Macerate the testes with a pair of clean forceps until clumps are no longer visible to the naked eye.
    
   
7. Add 2 mL of cold 1X NPB. Mix well by pipetting the solution up and down with a 10-mL plastic pipette.
    
   
8. Filter the sperm suspension through four thicknesses of cheesecloth placed into a funnel. Collect the filtrate in a 14-mL tube.
    
   
9. Rinse the Petri dish with an additional 3 mL of cold 1X NPB. Force this through the cheesecloth into the 14-mL tube.
    
   
10. Add 5 mL of cold 1X NPB to the funnel. Wearing gloves, squeeze the cheesecloth by hand to force any remaining liquid through the funnel into the 14-mL tube.
     
    
11. Pellet the sperm by centrifugation at 3000 rpm for 10 min at 4°C using a swinging bucket rotor with the appropriate adapters.
     
    
12. During the centrifugation, allow 1 mL of 1X NPB to equilibrate to room temperature.
     
    
13. Decant the supernatant obtained from Step 11. Resuspend the sperm in 9 mL of 1X NPB using a 10-mL plastic pipette.
     
    
14. Recentrifuge the sperm at 3000 rpm for 10 min at 4°C. Decant the supernatant.
     
    
15. Resuspend the pellet with a 1-mL pipette tip in the 1 mL of room-temperature 1X NPB (from Step 12).
     
    
16. Add 50 µL of freshly made 10 mg/mL lysolecithin. Mix gently. Incubate for 5 min at room temperature.
     
    
17. Add 10 mL of cold 1X NPB supplemented with 3% BSA to the suspension to stop the reaction.
     
    
18. Centrifuge the samples at 3000 rpm for 10 min at 4°C. Decant the supernatant.
     
    
19. Resuspend the pellet in 5 mL of cold 1X NPB supplemented with 0.3% BSA. Mix well by pipetting with a 5-mL plastic pipette.
     
    
20. Centrifuge at 3000 rpm for 10 min at 4°C. Decant the supernatant carefully.
     
    
21. Resuspend the pellet in 500 µL of SSB. Transfer the suspension into a 1.5-mL tube.
     
    
22. Count the number of sperm nuclei using a hemocytometer.
     
    For details on this procedure, please consult Estimation of Cell Number by Hemocytometry Counting.
     
    
    1. Cut off the end of a 200-µL pipette tip with a razor blade.
        
       
    2. Mix the sperm nuclei well by pipetting.
        
       
    3. Dilute 1 µL of the sperm nuclei with 100 µL of SDB.
        
       
    4. Add 1 µL of the diluted Hoechst reagent.
        
       
    5. Visualize the sperm nuclei under a fluorescence microscope.
        
       
    For a 1:100 dilution, we typically obtain counts of 100 x 10⁴ to 200 x 10⁴ nuclei/mL in a 1 x 1 x 0.1-mm square of an improved Neubauer hemocytometer. At this concentration, the undiluted stock contains 1x 10⁵ to 2 x 10⁵ nuclei/µL. See Troubleshooting.
     
    
23. For best cryopreservation of the sperm, leave fresh nuclei overnight at 4°C to allow the glycerol to penetrate.
     
    
24. The next day, divide the sperm into 20-µL aliquots. Fast-freeze the samples in liquid nitrogen. Store the frozen aliquots at -80°C.
     
    

• 1.0% agarose, prepared in 0.1X MMR
   
  
• Cysteine solution (1X MMR containing 2.5% L-cysteine hydrochloride 1-hydrate, pH 7.8-8.0), prepared immediately before use
   
  
• Ficoll
   
  
• 10 mg/mL gentamycin (1000X stock)
   
  
• High-speed egg extract (see Generation of Transgenic Xenopus laevis: I. High-Speed Preparation of Egg Extracts, above)
   
  
• 1000 U/mL human chorionic gonadotropin (HCG)
   
  
• 100 ng/µL linearized plasmid
   
  
• Any enzyme can be used for linearization of plasmid. Digest the DNA using standard conditions, and purify by phenol/chloroform extraction and ethanol precipitation. There is no need to gel-purify the plasmid.
   
  
• 100 mM MgCl₂
   
  
• 10X Marc's modified Ringer's (MMR), diluted to 1X prior to use and stored on ice
   
  
  • 20 mM CaCl₂
     
    
  • 50 mM HEPES (pH 7.5)
     
    
  • 20 mM KCl
     
    
  • 10 mM MgCl₂
     
    
  • 1 M NaCl
     
    
  • Adjust pH with NaOH to 7.5. Sterilize by autoclaving.
     
    
• 100 U/mL pregnant mare serum gonadotropin (PMSG)
   
  
• Restriction enzyme
   
  
• Sperm dilution buffer (SDB)
   
  
  • 75 mM KCl
     
    
  • 0.5 mM spermidine trihydrochloride
     
    
  • 0.2 mM spermine tetrahydrochloride
     
    
  • 250 mM sucrose
     
    
  • Add ~80 µL of 0.1 N NaOH per 20 mL of solution to titrate the pH to 7.3-7.5. Store 0.5-1 mL aliquots at -20°C.
     
    
• Sperm nuclei (1 x 105 to 2 x 105 nuclei/µL; see Generation of Transgenic Xenopus laevis: II. Sperm Nuclei Preparation, above)
   
  
• Xenopus laevis, adult females
   
  

1. Set the micropipette puller using the following conditions: p = 50, v = 100, and t = 5.
    
   
2. Use the puller to produce large transplantation needles with long, gently sloping tips.
    
   
3. Using the ocular micrometer from a dissecting microscope for measurement, clip the needles with a forceps to produce a beveled tip with a diameter of 80-100-µm (Fig. 1B ).
    
   

 
Figure 1. Nuclear transplantation apparatus. (A) This protocol uses an oil-filled injection system for the nuclear transplantations. The syringe and tubing are filled with mineral oil and the infusion pump depresses the syringe plunger, resulting in a constant, desirable flow rate. (B) The transplantation needle has a gently sloping tip and is clipped with forceps to produce a beveled 80-100-µm-wide tip.
 

4. Set up the transplantation apparatus (see Fig. 1A for details). Fill the plastic tubing and gas-tight syringe with mineral oil. Set the flow rate of the syringe pump at 0.6 µL/min. Start the infusion pump before injection to stabilize the flow.
    
   
Preparation of Transgenic Nuclei
 

5. Using a clipped 200-µL pipette tip, mix 4 µL of sperm nuclei (~4 x 105 to 8 x 105 nuclei) with 1-2 µL of linearized plasmid (100 ng/µL). Incubate for 5 min at room temperature.
    
   
6. Dilute 0.5 µL of the restriction enzyme of choice with 4.5 µL of H2O.
    
   
7. Mix 1 µL of the diluted enzyme with 18 µL of SDB, 2 µL of 100 mM MgCl₂, and 2 µL of high-speed egg extract.
    
   
8. Add the enzyme solution from Step 7 to the sperm/DNA mixture from Step 5. Mix well by gently pipetting using a clipped 200-µL pipette tip. Incubate for 15 min at room temperature.
    
   
Preparation of Injection Dishes
 
These dishes are designed for injection of transgenic nuclear material into Xenopus eggs (during Steps 16-26) and should be prepared in advance.
 

9. Pour the 1.0% agarose solution into 60-mm Petri dishes.
    
   
10. Before the agarose solidifies, lay a weighing boat onto each plate as a template.
     
    
11. After the agarose solidifies, remove the templates. Wrap the dishes in Parafilm, and store them at 4°C until use.
     
    
Preparation of Recipient Eggs
 

12. About 3-5 d prior to HCG injection, prime two adult female X. laevis by injecting 50 U of PMSG into the dorsal lymph sac using a 26-gauge needle fitted to a 1-mL syringe.
     
    
13. Twelve to fifteen hours before transplantation, inject each frog with 500 U of HCG. Store the frogs overnight at 15°C-18°C.
     
    
14. While the enzyme reaction is incubating (Step 8), manually expel the eggs from the frogs. Squeeze the eggs directly into a large dry beaker.
     
    
15. Add the cysteine solution immediately to the eggs to ensure egg quality. Dejelly the eggs in the cysteine solution.
     
    This usually takes ~10 min, so by the time the eggs are ready, the enzyme reaction (Step 8) is nearly complete.
     
    
16. Wash the dejellied eggs with 1X MMR at least three times. Use a wide-bore Pasteur pipette to transfer the eggs to injection dishes containing 0.4X MMR supplemented with 6% Ficoll and 10 µg/mL gentamycin. Fill the square space with eggs so that no gap is left between the eggs.
     
    
17. Place the injection dish on an empty plastic box half-filled with ice. Place the box and eggs under the injection microscope.
     
    This should provide a transplantation temperature of ~16°C.
     
    
Injection of Transgenic Nuclei
 

18. After incubation with the enzyme (Step 8), mix the sperm nuclei gently by pipetting with a clipped 200-µL pipette tip. Transfer 5 µL of the reaction into 150 µL of SDB equilibrated to room temperature.
     
    Keep decondensed sperm nuclei at room temperature and transplant them within 1 h, but preferably within 30 min.
     
    
19. Attach a piece of Tygon tubing to the end of a clipped 200-µL pipette tip (Fig. 2A ).
     
    

 
Figure 2. Loading the transplantation needle. (A) The reaction mix (containing diluted, restriction-enzyme-treated sperm nuclei) is drawn into a clipped, 200-µL, yellow pipette tip attached to ~0.5-1cm of Tygon tubing. (B) The 200-µL pipette tip containing the Tygon tubing and dilute sperm nuclei is carefully detached from the Pipetman and connected to the blunt end of the transplantation needle using the tubing. The needle is gently loaded with the dilute sperm nuclear reaction by gravity. This is done by slowly increasing the angle of the pipette tip/tubing/needle so that the mixture flows gently into the needle. Once the needle is completely filled, it is detached from the tubing and is ready to connect to the infusion pump. The remaining sperm mixture can be set aside horizontally and used to reload another needle, if two people are injecting simultaneously or if a needle is accidentally damaged or blocked.
 

20. Using this tip, mix the diluted sperm nuclei well, but avoid making bubbles. Fill the clipped 200-µL pipette tip with the diluted sperm suspension.
     
    
21. Carefully detach the clipped 200-µL pipette tip from the Pipetman, keeping the tip horizontal. Attach the end of the Tygon tubing opposite the tip to the blunt end of a transplantation needle.
     
    
22. Backfill the transplantation needle by slowly increasing the angle of the pipette tip/tubing/needle so that the mixture flows gently into the needle (Fig. 2B).
     
    You can keep the 200-µL pipette tip with the remaining nuclei by placing it horizontally, in case you need to load another needle.
     
    
23. Attach the needle to the mineral-oil-filled tube connected to the syringe in the syringe pump.
     
    
24. Check the flow on the pump and start injecting. Keep the needle inside each egg for ~0.5 sec. Move the needle fairly rapidly from egg to egg, piercing the plasma membrane of each egg with a single, sharp motion.
     
    We usually transplant for ~15-20 min. See Troubleshooting.
     
    
Incubation of Transgenic Embryos
 

25. After injection, transfer the embryos in their injection dishes to an incubator set at 16°C.
     
    
26. When the embryos reach the four-cell stage (about 3-4 h after injection at 16°C), use a wide-bore Pasteur pipette to transfer normally dividing embryos gently to a 10-cm Petri dish containing 0.1X MMR supplemented with 6% Ficoll and 10 µg/mL gentamycin.
     
    Alternatively, the embryos can be transferred in groups of up to four or so embryos per well into 24-well dishes.
     
    See Troubleshooting.
     
    
27. The next day (when embryos are around stage 12), transfer healthy embryos to a new 10-cm Petri dish (or 24-well dishes) containing 0.1X MMR supplemented with 10 µg/mL gentamycin.
     
    Because of the large needle tip used for transplantations, embryos often develop large blebs at the site of injection. These blebs occur when cells are forced out of the hole left in the vitelline membrane at the injection site. They generally do not affect development, falling off at the neurula or tailbud stages.
     
    See Troubleshooting.
     
    
28. Incubate embryos at 14°C-22°C until they reach the stage of interest for your specific experimental needs.
     
    See Troubleshooting.