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Hydrostatic Pressure Gynogenesis Protocol

Pressure Bomb Apparatus

DAY ONE

  1. Gather females of interest. One should have at least 6 females to insure that most of the females lay eggs.
  2. Prime females with HCG using a 1-cc syringe with a 28½ gauge needle in the afternoon- 10 I.U. HCG (Chorulon, Intervet Inc.) diluted with the accompanying diluent for a total volume of 100 uL. It is best to remove the hormone and diluent via syringe and place them in separate eppendorf tubes. Then measure the appropriate volumes of each with a pipet and mix the solution in a third eppendorf.
  3. The females are kept at 25oC overnight.

 

DAY TWO

  1. Boost females with HCG in the morning-100 I.U. HCG in a total volume of 100uL. It generally takes approximately 4 to 4.5 hours for the females to start producing eggs. The females may remain at room temperature during this time or they may be returned to 25oC.
  2. Gather males for sacrifice- 2 males for 4-5 frogs. Put the males in room temperature 1:500 MS222 (3-aminobenzoic acid ethyl ester methanesulfonate salt) at least 45 minutes prior to squeezing females.
  3. Make necessary solutions:

    1/10 MBS (1L)

    1/10 MBS + gentamycin (1L)

    1/20 MBS + 0.1% BSA (1L)

    1 x MBS + 0.1% BSA (50ml)

    10% BSA stock (keep frozen) (50ml)

    All the solutions are prepared as described in Early Development of Xenopus laevis: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2000)
  1. Flush the bomb with distilled water for at least five minutes.
  2. Coat 15mm x 60 mm petri dishes with 1 x MBS + 0.1% BSA.
  3. In 15 mL conical tube, add 2mL of 1 x MBS + 0.1% BSA; add 100 ul of 1 x MBS + 0.1% BSA to each of 2 eppendorf tubes.
  4. Dissect testes out of males once they have been in MS222 for at least 30 minutes. Dissect on ice to prevent frogs from waking up. Each pair of testes goes into its own eppendorf tube.
  5. Mash testes with sterile masher until homogenous. Transfer the volume of one eppendorf into the second to consolidate the sperm prep. Rinse the masher with 1 ml of 1 x MBS + 0.1% BSA into the eppendorf. Remove the sperm prep from the eppendorf and place into the 15 ml conical tube containing 1 x MBS + 0.1% BSA. Allow to settle for ~ 1 minute.
  6. Aliquot 250 ul of sperm into glazed 15 x 60 mm petri dishes. Start with the bomb dishes, then haploid dishes, and finally the controls. It is imperative that the bomb and haploid dishes do not contain any bubbles or small pieces of tissue. Place the dishes, uncovered , containing the bomb and haploid sperm into a UV cross linker (Fisher Biotech, model number FB-UVXL-1000) and irradiate with 1000 x 100 uJ/cm2 energy. DO NOT IRRADIATE CONTROL SPERM.
  7. Quickly, but carefully squeeze females' eggs into a clean, dry 15 x 100 mm petri dishes (unglazed). One petri dish per female. Avoid getting any water on the eggs because this will activate the eggs. The eggs must be used within 10 minutes to avoid egg death.
  8. Aliquot the females' eggs into each sperm-containing petri dish using a disposable plastic transfer pipette. The bomb should get the most eggs, the haploid dish gets the least amount of eggs. Be sure to glaze the pipette with 1 x MBS + 0.1% BSA to aliquot the eggs in order to avoid the eggs sticking to the inside of the pipette. Gently, but thoroughly, swirl the petri dishes and let sit 5 minutes to activate fertilization.
  9. Set the bomb up during this 5-minute incubation period.
  10. Fill reservoir about 1/3 the volume with 1/10 MBS. Remove any liquid from the top of the reservoir to insure a proper seal. Aliquot embryos into separate small glass vials.
  11. Fill the vials completely with 1/20 MBS + 0.1% BSA. Once the eggs are flooded, you have 5 minutes to get the bomb up to pressure. Place jars in the bomb reservoir, fill the reservoir completely with 1/10 MBS and secure the lid tightly. Fill the French chamber cell with 1/10 MBS until liquid is flush with bore opening. Insert T-shaped bar into the pressure cell. When liquid begins to come out of the tubing connected to reservoir, shut off the valve on the reservoir lid by turning it completely to the right (do not close it too tightly). Line up cylinder so it is directly centered under the hydraulic jack. Lock the jack by turning the screw above the jack to the right as far as it will go. Lower hydraulic jack onto the French chamber until it reaches a pressure of 3500-3700 psi. Embryos stay under pressure for 6 minutes.
  12. During this time, flood haploid and control embryos with 1/20 MBS + 0.1% BSA.
  13. After 6-minute incubation, release jack by turning screw above the jack all the way to the left. Release the pressure valve on the reservoir. Remove the lid of the reservoir. Transfer embryos to their own separate 15 x 60 mm petri dishes. Cleavage will begin in approximately 1.5 hours.
  14. Once the embryos have reached 4-cell stage, remove the media and add 2 % cysteine in 1/10 MBS (pH 7.9 + 0.1) to each petri dish and put on an orbital shaker for 6 minutes. Then remove cysteine and rinse each dish at least 5 times with 1/20 MBS + 0.1% BSA.
  15. Sort embryos into 15 x 100 mm petri dishes using a glass pipette, keeping all the cleavers and discarding dead or unfertilized embryos (100 embryos per dish). Embryos are kept at 22oC . The embryos are kept in 1/10 MBS + gentamycin.

DAY THREE & FOUR

  1. Sort embryos with a glass pipette, discarding any dead ones. Remove media and replace with fresh 1/10 MBS + gentamycin. Note any significant appearances or defects including gastrulation defects.

DAY FIVE

  1. Sort, but change media to dechlorinated water with a small amount of sera micron. The tadpoles should be fed starting at about stage 40-41. The amount of sera micron is critical; overfeeding can cause a bacterial or fungal bloom in the dishes. The mixture of water and sera micron should have an “imaginary” tint of green. If mixture is noticeably green, too much sera micron is present. (See section on “Animal Husbandry” for information on sera micron and tadpole feeding). Note any significant defects.

DAY SIX

  1. Anesthetize tadpoles using a fresh, sterile aliquot of 1:500 MS222. Apply 10 to 20 drops of MS222 to the dish to anesthetize the tadpoles. Score tadpoles on mutant phenotype. Be sure to transfer the tadpoles to water if you want them to wake up.

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Last update: Feb. 13, 2008