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Genetics: transgenics

3. Sperm Nuclei Preparation (or go back to 2. extract preparation)

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.
• 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 15 mM the pH is 7.7, should require about 5.5 mls of 10 N KOH for 100 mls; filter sterilise and store 0.75 ml aliquots at -20°C) Note: Dilution drastically changes the pH of HEPES, making it impossible to pH the stock directly.
• 10 mM spermidine trihydrochloride (filter sterilise and store 2.5 ml aliquots at -20°C)
• 10 mM spermine tetrahydrochloride (filter sterilise and store 1 ml aliquots at -20°C)
• 100 mM Dithiothreitol (filter sterilise and store 0.5 ml aliquots at -20°C)
• 500 mM EDTA pH 8 (stored at room temperature)
• 10% Bovine Serum Albumin (BSA; fraction V), (in H2 0; store 3.5 ml aliquots at -20°C)
• 10 mg/ml leupeptin (Boeringer Mannheim 1017 101; in DMSO; store 40 ml aliquots at -20°C.)
• 0.3 M phenylmethylsulfonyl fluoride (PMSF) (in EtOH, store at -20°C)
• 100% glycerol (sterilise by autoclaving and store at room temperature)
• 10 mg/ml Hoechst No. 33342 (in dH2 0; store in a light-tight vessel at -20°C)
• Sperm Dilution Buffer (SDB) (250 mM sucrose; 75 mM KCl; 0.5 mM spermidine trihydrochloride; 0.2 mM spermine tetrahydrochloride; add about 80µl of 0.1N NaOH per 20mls solution to titrate to pH 7.3-7.5; store 1 ml aliquots at -20°C.)

B. Solutions for Sperm Nuclear Preparation.

On the day of the sperm nuclei preparation, make up the following

• 50 ml of 1X MMR 45 ml H2 0 5 ml 10X MMR store on ice
• 25 ml of 2X Nuclear Preparation Buffer (NPB) 2X Final concentration 11.8 ml H2 0 8.33 ml 1.5 M sucrose 500 mM 0.75 ml 1 M HEPES 30 mM 2.5 ml spermidine trihydrochloride 1 mM 1 ml spermine tetrahydrochloride 0.4 mM 0.5 ml 100mM Dithiothreitol 2 mM 0.1 ml 500mM EDTA 2 mM store on ice
• 30 ml of 1X NPB w/ protease inhibitors 15 ml H2 0 15 ml 2X NPB 30 ul 10mg/ml leupeptin (added immediately before use) 30 ul 0.3 M PMSF (added immediately before use) store on ice
• 100 ml of 10 mg/ml L-a-lysophosphatidylcholine (Lysolecithin) (Sigma L-4129 or CalBiochem 440154); dissolve in H2 0 at room temperature immediately before use. Store solid stock desiccated at -20°C. Discard the stock powder if it becomes sticky.
• 10 ml of 1X NPB + 3% BSA w/ protease inhibitors 2 ml H2 0 5 ml 2X NPB 3 ml 10 % BSA 10 ul 10mg/ml leupeptin (added immediately before use) 10 ul 0.3 M PMSF (added immediately before use) store on ice
• 5 ml of 1X NPB + 0.3% BSA 2.35 ml H2 0 2.5 ml 2X NPB 0.15 ml 10 % BSA store on ice
• 0.5 ml 1X NPB + 30% glycerol + 0.3% BSA (Sperm Storage Buffer) 85 ul H2 0 250 ul 2X NPB 15 ul 10 % BSA 150 ul 100 % glycerol store on ice

C Sperm Nuclei Preparation ( largely based on Murray, 1991).

1) Anaesthetise a male by immersion in 0.1% aminobenzoic acid ethyl ester (Tricaine; MS222) (Sigma A-5040) with 0.1% sodium bicarbonate for at least 20 minutes and then pith it. Cut through the ventral body wall and musculature and lift the yellow fat bodies to isolate the two testes which are attached to the base of the fat bodies one on each side of the midline. Remove the testes with dissecting scissors and place them in a 35mm tissue culture dish containing cold 1X MMR. We routinely isolate sperm nuclei from one testis and use the other for in vitro fertilisations. Inspect the two testes and isolate the one with less blood contamination for the nuclear prep. (We store the other testis in 1X MMR, 10% fetal calf serum, and gentamycin in a vial at 4 ¡C and use it for in vitro fertilisations for up to a week.) If a large prep is required, testes from two to four males can be used. The final resuspension volume should be increased accordingly.

2) Rinse the testis in three changes of cold 1X MMR.

3) Using fine forceps, remove any remaining fat body and excess blood. Do not try to remove the blood vessels. Rather, puncture holes in the largest vessels and gently push the blood out. Take care not to puncture the testis as this releases sperm. 2) Place the cleaned testis in another 35mm tissue culture dish with 5 ml of cold 1X NPB + protease inhibitors for 2 to 5 minutes. 3) Transfer the testis to a dry 35mm tissue culture dish, and macerate the tissue well (until clumps are no longer visible to the naked eye) with a pair of clean forceps.

4) Resuspend the macerated testes in 2 mls of cold 1X NPB + protease inhibitors by pipetting the mixture up and down through a sterile, disposable 5 ml pipette.

5) Squirt the sperm suspension through two-four thicknesses of cheesecloth placed into a funnel and collect the solution into a 14 ml sterile culture tube (Falcon 2059; 17 x 100 mm). Rinse the dish with an additional 3 ml of cold 1XNPB + protease inhibitors and add to the cheesecloth. After adding 5 ml more (10 mls total) of cold 1XNPB + protease inhibitors use a gloved hand to fold the cheesecloth and squeeze any remaining liquid through the funnel into the 14 ml tube. We usually end up with 9 ml of sperm suspension in the tube.

6) Centrifuge the sperm suspension at 3,000 rpm for 10 min. at 4°C (we use a Sorvall HB-4 or similar swinging bucket rotor fitted with the appropriate adapters). The sperm pellet should be white, fairly compact. Usually we have some blood contamination which can be seen in the center of the pellet. During the spin, allow 1 ml of 1XNPB + protease inhibitors to equilibrate to room temperature.

7) Decant the supernatant and resuspend the sperm pellet in 9 ml of cold 1XNPB + protease inhibitors and repellet by centrifugation at 3,000 rpm, 10 min., 4°C. During this spin dissolve 1 mg of L-a-lysophosphatidylcholine (Lysolecithin) (Sigma L-4129) in 100 ul (10mg/ml) of H2 0 at room temperature. Lysolecithin will not remain in solution below room temperature.

8) Decant the supernatant and resuspend the sperm pellet in the 1 ml of 1XNPB + protease inhibitors that has equilibrate at room temperature and add 50µl of 10mg/ml lysolecithin. Mix gently and incubate for 5 minutes at room temperature.

9) Add 10 ml cold 1XNPB +3% BSA + protease inhibitors to the suspension and centrifuge at 3,000 rpm, 10 min., 4°C. At the end of this spin the pellet should now be wider and more loose than before. In addition the pellet should no longer have redness. The looseness and the loss of haemaglobin mean that the pellet now contains nuclei rather than intact cells.

10) Decant the supernatant and resuspend the pellet in 5 ml cold 1XNPB + 0.3% BSA (no protease inhibitors), mix gently by pipetting up and down, and centrifuge at 3,000 rpm, 10 min., 4°C.

11) Decant the supernatant and resuspend the pellet in 250µl of 1XNPB + 30% (w/v) glycerol + 0.3% BSA (Sperm Storage Buffer) and transfer suspension into a 1.5 ml eppendorf tube. Store at 4¡C and use for transgenesis for up to 48 hours.

12) Cut the tip of a yellow tip with a razor blade and mix the sperm nuclei suspension by pipetting up and down. Remove 2 µl and dilute into 200 ul of sperm dilution buffer (i.e. 1:100 dilution). Add 2 µl of a 1:100 diluted Hoechst stock and transfer the diluted sperm nuclei to a hemacytometer for counting. Visualise the sperm nuclei under a fluorescence microscope using a DAPI/Hoechst filter set. For a 1:100 dilution of our sperm nuclei stock, we typically obtain counts of 125-200 (X10 4 nuclei/ml). At this concentration, the undiluted stock contains 125-200 nuclei/nl. If your sperm stock is substantially less concentrated (i.e.. a count of <100 for a 1:100 dilution), repellet the sperm nuclei at low speed (or allow the nuclei to settle over a few hours) and resuspend in a smaller volume of sperm storage buffer. We store the fresh nuclei overnight at 4¡C and after extensive mixing by pipetting up and down with a cut yellow tip, we freeze 40 µl aliquots in liquid nitrogen and store the frozen nuclei at -80°C. One aliquot is thawed for each day of transgenesis.

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