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Genetics: transgenics

2. High Speed Extract Preparation (or go back to 1. introduction)

2.a Stock solutions

2.b Additional solutions

2.c Making the extract

A. Stock Solutions:

• 10X Marc's Modified Ringer's (MMR) (1 M NaCl; 20 mM KCl; 10 mM MgCl2 ; 20 mM CaCl2 ; 50 mM HEPES, pH 7.5) (Sterilise by autoclaving and store at room temperature.
• 20X Extract Buffer (XB) salts (2M KCl; 20 mM MgCl2 ; 2 mM CaCl2 ; filter sterilise and store at 4°C
• 1.5 M Sucrose (filter sterilise and store 8.33 ml aliquots at -20°C)
• 1 M HEPES (titrate with KOH so that when diluted to 10 mM the pH is 7.7, should require that the pH of the 1M stock is around 9.5; filter sterilise and store 0.75ml aliquots at -20°C) Note: Dilution drastically changes the pH of HEPES.
• 1M CaCl2 (filter sterilise and store at 4°C)
• 1M MgCl2 (filter sterilise and store at room temperature)
• 200mM EGTA (adjust pH to 7.7, sterilise by autoclaving and store at room temp.)
• Versilube F-50. Made by General Electric. Can be purchased from Andpak-EMA [1560 Dobbin Drive, San Jose, CA 95133; Tel. (408)-272-8007].
• Energy Mix (150 mM creatine phosphate (Boeringer Mannheim Biochemicals 127 574; 20 mM ATP (Boeringer Mannheim Biochemicals 519 979; 20 mM MgCl2 ; made up in H2 0; store 0.1 ml aliquots at -20°C)
• 100 U/ml Pregnant mare serum gonadotropin (PMSG; 367222; Calbiochem; made up in sterile H2 0; store 10 ml aliquots at -20°C.)
• 1,000 U/ml Human chorionic gonadotropin (Sigma CG-10); made up in sterile H2 0; stored at 4°C.
• 10 mg/ml leupeptin (Boeringer Mannheim 1017 101; in DMSO; store 40 ml aliquots at -20°C.)
• 10 mg/ml chymostatin (Boeringer Mannheim 1 004 638) and pepstatin (Boeringer Mannheim 600 160); in DMSO; store 40 ml aliquots at -20°C.)

B. Additional Solutions for High Speed Extract Preparation.

On the day of the extract preparation, make up the following:

• 4000 ml 1X MMR
• 3600 ml H2 0
• 400 ml 10X MMR
• 1000 ml 2% (w/v) cysteine in 1X XB salts 900 ml H2 0 50 ml 20 XB salts 20g cysteine (Sigma C-7755) titrate to pH 7.8 with 5N NaOH. Make up within 1 hour of use.
• 500 ml Extract Buffer (XB) 450 ml H2 0 Final concentration 25 ml 20X XB salts 100mM KCl; 1mM MgCl2 ; 0.1 mM CaCl2
• 16.7 ml 1.5M sucrose 50mM Sucrose 5 ml 1M HEPES 10mM HEPES, pH 7.7
• 75 ml CSF-XB 66 ml H20 Final concentration 3.75 ml 20X XB salts 100mM KCl; 2mM MgCl2; 0.1 mM CaCl2 75 ml of 1M MgCl2 2.5 ml 1.5M sucrose 50mM Sucrose 0.75 ml 1M HEPES 10mM HEPES, pH 7.7 1.88 ml 200mM EGTA, pH 7.7 5mM EGTA 75 ml 10 mg/ml chymostatin/pepstatin mix 10 mg/ml 75 ml 10mg/ml leupeptin 10 mg/ml

C. High Speed Extract Preparation

The high speed extract preparation is largely based on Murray (1991). Briefly, a crude cytostatic factor (CSF) arrested egg extract (cytoplasm arrested in meiotic metaphase) is prepared. Calcium is added to drive the extract into interphase and a high speed spin is performed to obtain a purer cytoplasmic fraction. Cytochalasin is omitted because it interferes with normal development. Although a crude extract can be used for making transgenic embryos, high speed extracts have several advantages over crude extracts.

• High speed extracts promote decondensation of sperm nuclei but do not promote DNA replication
• high speed extracts cannot progress through the cell cycle
• high speed extract can be stored frozen (at -80°C) for many months before use
• sperm nuclei are more easily mixed in high speed extracts which do not contain actin polymers.

1) Prime 8-12 female adult X. laevis about 48 hours prior to HCG injection by injecting 50U of pregnant mare serum gonadotropin (PMSG) into the dorsal lymph sac. Maintain at room temperature. The evening before the extract preparation, inject each frog with 500U HCG and place two frogs per container into 2 liters 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 18-19°C overnight (12-14 hours).

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 which 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; optimally, the high speed spin should begin within 45-60 minutes of dejellying the eggs.

3) Overnight the frogs should have laid eggs into the 2 liters of 1X MMR. The high salt in 1X MMR should keep the eggs unactivated and healthy. Manually expel any remaining eggs from each frog into the 2 liter of 1X MMR. Remove the frogs and transfer the eggs from each container into separate 500ml beakers containing around 50ml of 1X MMR. If the eggs look unhealthy, eliminate the eggs from the prep. 3) Remove as much MMR as possible from the eggs. Dejelly eggs in 2% cysteine in XB salts (no HEPES/sucrose) at pH 7.8. Add a small amount at a time, swirl eggs, and partially replace with fresh cysteine several times during dejellying. Dejellying should be performed separately for different batches of eggs and batches which show breakage or egg activation during dejellying should be discarded.

4) Once the eggs from all the batches have been dejellied, the remaining good eggs are pooled and washed thoroughly in XB (with HEPES/sucrose). We use about 125 mls for each wash and do 4 washes.

5) Wash eggs in CSF-XB with protease inhibitors. We do two 35 ml washes.

6) Using a wide-bore pasteur pipette, transfer eggs into Beckman ultraclear tubes. For these volumes, we typically use 14X95 mm tubes (Catalog #: 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 seconds at 1000 rpm (150 g) and then 30 seconds at 2000 rpm (600 g). 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 minutes at 10,000 rpm 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 gauge 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 greyish rather than golden at this step. After a second spin to clarify this extract, it should be golden.

9) Estimate the volume of extracts and add the appropriate amount of protease inhibitors to the isolated cytoplasm (do not add cytochalasin); recentrifuge the cytoplasm in Beckman tubes for an additional 10 minutes at 10,000 rpm to clarify, again using a swinging bucket rotor. Collect the clarified cytoplasm as before. Expect to get about 0.75-1 ml cytoplasm per batch of eggs collected from one frog.

10) Add 1/20th volume of the ATP-regenerating system (Energy Mix). Transfer the clarified cytoplasm into TL100 tubes thick wall polycarbonate tubes (Beckman 349622). Tubes hold about 4 mls each and should be at least half full.

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

12) Spin tubes in a Beckman tabletop TL-100 ultracentrifuge in a TL100 rotor (fixed angle) at 70,000 rpm for 1.5 hours 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 was 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 15 µl aliquots in 0.5ml eppendorf tubes. Quick freeze aliquots in liquid nitrogen and store at -80¡C until use. We typically obtain 2-3 ml of high speed cytosol from preparations of this scale. Sperm nuclei should be incubated in an aliquot of extract and stained with Hoechst to determine whether extract is active in decondensation. Active interphase extract should visibly swell sperm nuclei (thicken and lengthen) within 10-15 minutes of addition to extract at room temperature.

Next: 3. Sperm Nuclei Preparation

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