(Hajime Ogino, 3/16/2004, modified 4/24/07)
We have modified the original protocol developed by Kroll and Amaya (Development 122, 3173-3183, 1996) to improve efficiency. Consult our “previous version” of the transgenic protocol for general background and details of the method, published in Early Development of Xenopus laevis: A Laboratory Manual (Sive, H. L., Grainger, R. M. and Harland, R. M., Cold Spring Harbor Laboratory Press, 2000). The protocol described here is the latest version of our protocol that includes both the modifications originally made for transgenesis in X. tropicalis (Offield, M. F. et al., Development 127, 1789-1797, 2000, Hirsh, N. et al., Dev Dyn. 225, 422-433, 2002) and some further refinements. The efficiency of transgenesis with this protocol is around 10%: ~30% of injected eggs cleave normally, ~70% of normal cleavers gastrulate normally, and ~50% of the remaining normally gastrulated embryos express a transgene. The major changes from previous iterations of the protocol are as follows:
1) Use of oocyte extract instead of egg extract. This modification was originally made for the transgenic procedure in X. tropicalis (Hirsh, N. et al. Dev Dyn. 225, 422-433, 2002), but also works well in X. laevis.
2) Use of MOH buffer instead of SDB for dilution of the sperm nuclei reactions (Offield, M. F. et al. Development 127, 1789-1797, 2000).
3) The restriction enzyme and MgCl2 may be omitted from the transgenic reaction mix. While the use of restriction enzyme stimulates transgene integration, it can also damage the host genome. Embryos carrying the damaged genome often develop abnormally and will show ectopic transgene expression. If the transgene is shorter than 5 kb, the restriction enzyme and MgCl2 can be omitted from the reaction. However, if the transgene is longer than 10 kb, the restriction enzyme and MgCl2 might be necessary to get consistent results.
4) 0.5% BSA is added to all solutions used after dejellying (i.e. 1x MMR for the post-dejellying rinse, the injection medium). Following dejellying, the unfertilized eggs are easily activated by mechanical stimuli—the BSA prevents the egg activation that often happens during the post-dejellying rinse and egg loading into the injection dishes. Omitting calcium from the dejellying and post-dejellying rinse solutions also helps to prevent the egg activation.
In addition, the BSA appears to maintain egg activity for fertilization prior to injection and aid in the healing of injected eggs. The underlying mechanisms are unclear, but the addition of BSA to the culture medium increases the number of transgenic embryos that gastrulate normally.
5) The plasmid backbones are fully removed from transgene constructs (Hirsh, N. et al., Dev Dyn. 225, 422-433, 2002). In the original Kroll and Amaya protocols, plasmids carrying a transgene construct are linearized and used directly in the transgenic reaction. However, we have found that the flanking vector sequence introduced with a transgene can interfere with correct expression of the transgene. This appears to be enhancer/promoter-specific, but in severe cases, strong background expression can mask the “correct” expression pattern of the transgene Thus, we completely remove the plasmid backbone sequences from a transgene cassette by restriction enzyme digestion followed by gel electrophoresis. The transgene is then purified from the agarose gel using the Qiaquick Gel Extraction kit (Qiagen), precipitated with ethanol once, and finally dissolved in 10 mM KCl when it is ready for use in transgenesis.
STOCKS AND PREMADE BUFFERS
I. Stock Media
add water to 1 Liter, pH to 7.4-7.6 with NaOH
Autoclave if made from dry stocks. Otherwise just combine sterile solutions + sterile H2O in a sterile bottle.
10x MMR (No Ca2+)
Prepare as above, but omit 20 mM CaCl2. This reagent is used in steps prior to injection to prevent egg activation.
pH to 7.4-7.6 with NaOH, autoclave for sterilization
II. Sperm Nuclei Prep
·1.5 M Sucrose (Sigma S-0389)
Dissolve 513.45 g in 1000 ml of deionized nanopure water, filter sterilize, and store in 11 ml aliquots at -20°C.
·1 M HEPES (Sigma H-8091) Dissolve 21.45 g in 90 ml deionized water. pH to 7.7, filter sterilize and store in 990 µl aliquots at -20°C.
Dissolve 21.45 g in 90 ml deionized water. pH to 7.7, filter sterilize and store in 990 µl aliquots at -20°C.
·100 mM spermidine trihydrochloride (Sigma S-2501)
Dissolve 0.255g in 10 ml of sterile water. Store in aliquots of 330 µl at -20°C.
·100 mM spermine tetrahydrochloride(Sigma S-1141)
Dissolve 1.74g in 50 ml of sterile water. Store in 140 µl aliquots at -20°C.
·1 M dithiothreitol (DTT) (Sigma D-9779)
Dissolve 1.54g in 10 ml of sterile water. Store in aliquots of 66 µl at -20°C.
·10% bovine serum albumin (BSA)(Sigma A-3294)
Slowly dissolve 40 g by floating it on 375 ml of deionized water. It should slowly go into solution. Bring final volume to 400 ml and pH to 7.6 with KOH. Final solution should look yellowish. Store at -20°C in 4 ml aliquots.
·10 mg/ml leupeptin (Roche 1 017 128)
Dissolve 10 mg in 1 ml of Dimethyl Sulfoxide (DMSO) (Sigma D-8418) and store in 14 µl aliquots at -20°C.
·0.3 M phenylmethylsulfonyl fluoride (PMSF) (Boehringer Mannheim 837 091)
Dissolve 78 mg in 1.5 ml of ethanol and store in 14 µl aliquots at -20°C.
·0.5 M EDTA (J.T. Baker 8991-01)
Dissolve 4.38g in 20 ml sterile water (Bring pH to 8.0 to dissolve). Bring final volume to 30 ml with sterile water. Sterilize and store at room temperature.
·10 mg/ml Hoechst No. 33342 (Sigma B-2261)
Dissolve 25 mg in 2.5 ml of distilled water. Wrap in aluminum foil and store at -20°C.
·100% Glycerol (glycerin) (Fisher G33-500)
·Digitonin (Calbiochem 300410)
Dissolve 10 mg in 1 ml of DMSO (Sigma D-8418). Store in 12 µl aliquots at -20°C.
·MS222 (3-Aminobenzoic Acid Ethyl Ester Methanesulfonate Salt; Sigma A-5040)
For the "anesthetizing bath" make up at a concentration of 1:500 in distilled water plus 1g/L Instant Ocean salts and 1g/L Sodium Bicarbonate. You can also make it up in 1/10x MBS. Adjust pH to 7.4-7.6 and store in 4°C.
III. Oocyte extract prep
·1.5 M Sucrose
·1 M CaCl2
·Collagenase A (Roche 1 088 793 or Sigma C-0130)
·0.1 M EGTA (USB 15703)
Dissolve 3.8 g in 100 ml of sterile water. Store at room temperature.
·10 mg/ml pepstatin A (Calbiochem 516482)
Dissolve 10 mg in 1 ml of DMSO. Store at -20°C.
·10 mg/ml leupeptin (Roche 1 017 128)
Dissolve 10 mg in 1 ml of DMSO (Sigma D-8418). Store at -20°C.
·10 mg/ml chymostatin (Roche 1 004 638)
Dissolve 10 mg in 1 ml of ethanol. Store at -20°C.
IV. Transgenesis reaction
0.7 ml 1 M CaCl2
100 ml 10x MBS salts
4 ml 5 M NaCl
888 ml water
pH to 7.4-7.6 with NaOH
Sperm Dilution Buffer 100 ml
250 mM Sucrose 16.7 ml 1.5 M Sucrose
75 mM KCl 7.5 ml 1 M KCl
0.5 mM Spermidine 0.5 ml 100 mM Spermidine
0.25 mM Spermine 0.2 ml 100 mM Spermine
2 mM HEPES 0.2 ml 1 M HEPES
Add NaOH to adjust pH to 7.4-7.6
Sterilize by filtration and store in 1-1.5 ml aliquots at -20°C.
MOH Injection Buffer
10 mM K-PO4
(100mM K-PO4 (pH 7.2) is made by mixing 28.3 ml 1M KH2PO4 and 71.7 ml 1M K2HPO4)
125 mM potassium gluconate
5 mM NaCl
0.5 mM MgCl2
250 mM Sucrose
0.25 mM Spermidine
0.125 mM Spermine
Add KOH to adjust pH to 7.2 if necessary.
Sterilize by filtration and store in 1-1.5 ml aliquots at -20°C.
100 mM MgCl2
Use in REMI reaction if you use a restriction enzyme.
1 x MMR (with Ca2+) (1 L)
10 x MMR (with Ca2+) 100 ml
distilled water 900 ml
pH to 7.5 with NaOH. Sterilize by autoclaving or use sterile water for the preparation. Store at RT. Use for preparing the injection medium and agarose-coated injection dishes.
10 % BSA (100 ml)
Dissolve 10g of BSA in 100 ml of distilled water. Sterilize by filtration and store at –20°C. Use for preparing the followings: post-dejellying rinse solution, injection medium, and culture medium.
Injection medium (100 ml)
1 x MMR (with Ca2+) 40 ml
Ficoll 6.0 g
10% BSA 5.0 ml
Fill to 100 ml with distilled water. Store at –20°C.
V. Other stocks/supplies information
·Ficoll 400 (Sigma F-4375)
·human chorionic gonadotropin (hCG) (“CHORULON” from INTERVET INC.)
·Gentamycin (cellgro No.61-098-RF) Make 50 mg/ml solution with distilled water.
·Glass Capillary Tubes (World Precision Instruments TW100-4)
·Dimethyldichlorosilane (Sigma D3879) (If you prefer siliconizing injection needles)
·Ultracentrifuge Tubes, open-top, polycarbonate 1/2" x 2" (Beckman 349622)
·Hamilton Gas-tight Syringes, 25 µl
·Insulin syringes for injecting HCG (B-D 33239420)
·Mineral Oil (Sigma M-8410)
·Pump (Harvard Apparatus 11 Syringe Pump, single or dual syringe)
·Tygon Tubing, ID 1/32", OD 3/32", wall 1/32" (Fisher 14-169-1A)
SPERM NUCLEI PREP
Preparation of sperm prep working solutions (to be made fresh each time & kept on ice)
2 X NPB(30 ml)
Adjust pH to 7.7 if necessary (use KOH).
1 X NPB (40 ml)
20 ml 2X NPB
20 ml sterile H20
1 X NPB + 3% BSA (12 ml)
6 ml 2 X NPB
2.4 ml sterile H20
3.6 ml 10% BSA
1 X NPB + 0.3%BSA (6 ml)
3 ml 2 X NPB
2.8 ml sterile H2O
180 µl 10% BSA
Sperm Nuclei Storage Buffer (1 ml)
500 µl 2 X NPB
300 µl glycerol
170 µl sterile H20
30 µl 10% BSA
1 x MMR (50 ml)
5.0 ml 10 x MMR (with Ca)
45 ml distilled water
Sperm nuclei preparation protocol
1) Anesthetize a male in 1:500 MS222 at room temperature (30-45 min). Make the above working solutions from the thawed components and place on ice.
2) After the frog is immobilized (about 30-45 minutes), remove two testes and place them into a 60 x 15 mm petri dish filled with ice-cold 1X MMR.
3) Clean testes (remove as much fat bodies and blood as possible - carefully poke holes in blood vessels and push blood out) under dissecting scope and rinse two times in ice-cold 1X MMR and two times in ice-cold 1X NPB. Clean as much as possible without damaging the testes and releasing sperm.
4) Put the cleaned testis in another dry petri dish and under a dissecting scope macerate the tissue well (until clumps are no longer visible to the naked eye) with two pairs of clean forceps. This can be done by gently grasping the testis with one pair of forceps and slowly teasing the other end with another pair of forceps. The entire macerating process should take around 15 minutes.
5) Resuspend the macerated testes in 2 ml of 1X NPB by gently pipetting the solution up and down through a fire-polished, truncated pasteur pipette or truncated plastic transfer pipette with an opening of about 3 mm in diameter. Next, gently pipette the solution up and down through a cut yellow pipette tip. Finally, use a regular yellow pipette tip to completely eliminate any aggregates.
6) Cut the snap cap from a 12 x 75 round bottom polypropylene tube (Falcon 2063) along its equator to make a ring of pastic.
Place the 2 x 2 cm square of Nitex mesh (100-110 µm, Nitex HC-3-110 etc.) across the opening of a 14 ml (17 x 100 mm) round bottom polypropylene tube (Falcon 2059). Secure the mesh by placing the plastic ring onto the mesh square and forcing it into the inner diameter of the tube.
Pass the sperm suspension through the mesh and into the tube using a pipettor with a cut blue pipette tip (we use round bottom polypropylene tubes; Falcon 2059). Rinse the forceps and dish with 8 ml of 1X NPB and force this through the mesh also. The sperm suspension may not flow through the homemade filter apparatus because of surface tension. Gently touch the mesh surface with the cut blue pipette tip to initiate the filtering.
7) Pellet the sperm by centrifugation at 3,000 rpm for 10 min. at 4°C (Sorvall HB-6 rotor). Don't be concerned if the pellet looks slightly reddish. Carefully aspirate off the supernatant without allowing the sperm pellet to come in contact with the air. Resuspend the sperm in 0.5 ml of 1 x NPB first by pipetting with the cut-wide and cut-narrow yellow tips. Then dilute to 8 ml NPB and repellet by centrifugation at 3,000 rpm, 10 min., 4°C.
8) Resuspend the pellet in 1 ml NPB with a cut plastic blue pipette tip, again not allowing the pellet to be exposed to air. Warm the suspension to room temperature.
9) Add 10 µl of Digitonin solution (10 mg/ml in DMSO) to the resuspended sperm pellet and incubate at room temperature for 5 minutes.
10) Add 12 µl each of the leupeptin and PMSF stock solutions to the cold 1X NPB +3%BSA solution. Mix gently by inversion.
11) Add 10 ml cold 1X NPB +3%BSA (with protease inhibitors) to the suspension, mix gently by inversion, and centrifuge at 3,000 rpm, 10 min., 4°C. Decant the supernatant.
12) Resuspend the pellet in 5 ml cold 1X NPB +0.3%BSA (no protease inhibitors), mix gently by inversion, and repellet as before.
13) Resuspend the pellet in 500µl sperm nuclei storage buffer using cut-off tips and transfer suspension into a 1.5 ml eppendorf tube. Count the sperm nuclei density using a hemacytometer (Fisher, cat. #: 02-671-5): dilute a small amount of the concentrated nuclei 1:100 in sperm dilution buffer and add 1µl of 1:100 Hoechst stock to visualize the sperm heads under a fluorescence microscope. For a 1:100 dilution of our sperm nuclei stock, we typically obtain counts of 75-125 (X104 cells/ml). At this concentration, the undiluted stock contains 75-125 sperm nuclei/nl. If your sperm nuclei stock is substantially less concentrated (i.e. a count of <50 for a 1:100 dilution), repellet the nuclei and resuspend in a smaller volume of sperm nuclei storage buffer. Sperm nuclei can be stored at 4°C and used for transplantations for up to 48 hours. The remained sperm nuclei can be stored at –80°C. Freshly prepared sperm nuclei are better, but the frozen nuclei also give satisfactory results. You can simply “slow freeze” the nuclei; ie-simply put them in the –80°C freezer. No need for liquid nitrogen, etc.
OOCYTE EXTRACT PREP
Preparation of oocyte extract prep working solutions
50% Leibowitz L-15 3.7 g Leibowitz L-15
15 mM HEPES 7.5 ml 1 M HEPES
+ gentamycin 500 µl 50 mg/ ml gentamycin
20 x XB Salts (250 ml)
from stocks from chemicals
2 M KCl ----- 37.28 g KCl
20 mM MgCl2 5 ml 1 M MgCl2 1.02 g MgCl2·6H2O
2 mM CaCl2 0.5 ml 1 M CaCl2 0.07 g CaCl2·2H2O
Sterilize by filtration.
1 x XB(1 L)
pH with KOH to pH 7.7
pH to 7.7
**Add 1:1000 of 10 mg/ml stock solutions of Leupeptin, Pepstatin, Chymostatin just before use. Use gloves when handling protease inhibitors.
Oocyte extract preparation protocol
1) Sacrifice 2 or 3 females and remove the entire ovaries. If oocytes are mostly large and there are a lot, use only 2 females. If one or both females have a lot of tiny oocytes or not many, use an extra female. It is very important to have enough starting material for this prep!
2) Cut ovaries into small pieces using scissors and digest for approximately 1.5-3.5 hours at 28°C (in water bath with agitation) in 3 mg/ml collagenase made up in OR2. It takes less time to complete the digestion if you split the oocytes into a few containers with approximately 100-150 ml of collagenase+OR2 solution in each container.
3) Begin checking the oocytes every 15 minutes after about an hour-they'll be ready once you no longer see any clumps of oocytes.
4) After digestion, wash the oocytes several times in OR2-if you swirl them in a flask, the small immature oocytes will float to the top and you can easily pour some of them off.
5) Transfer oocytes to OR3. The oocytes will keep in OR3 for about a week at 16-18°C. Leave overnight and sort the next morning. You can also begin the sorting process by removing any dead, dying, or tiny oocytes to save time for the next day.
II. Extract prep
1) Remove as many dead, dying, or tiny oocytes as possible or use a transfer pipette with a cut-off tip to transfer good oocytes into a separate dish. This is the step that requires a lot of time and a lot of work. You have to work quickly, and it is not necessary to remove every single undesired oocyte. You should probably try to be completely finished with your sorting by noon at the latest.
2) Rinse oocytes four times in 1 x XB (approx. 100 ml each rinse)
3) Rinse oocytes two times in CSF-XB with protease inhibitors added just before use. (approx. 50 ml each rinse).
4) Load oocytes gently into 30 ml centrifuge tubes (open-top corex tube) using a cutoff transfer pipet. Remove as much CSF-XB as possible and try to balance tubes visually, with approximately equal volumes in each centrifuge tube. (You should have at least enough oocytes to half fill two 30 ml centrifuge tubes. It is probably better if you start with enough to fill each tube 3/4, depending on the quality of the oocytes.)
5) Add 1 ml Versilube F-50 on top of oocytes in each centrifuge tube. Versilube F-50 is trademarked by General Electric, and is same with MIL-S-81087C (F-50) which can be purchased from Andpak-EMA.
6) Spin tubes in a clinical centrifuge at room temperature for about 60 seconds at 1000 rpm (150g), then for an additional 30 seconds at 2000 rpm (600g). This will pack the oocytes, but they should not be lysed or broken.
7) Remove the excess CSF-XB and Versilube, which should cover the oocytes after the packing spin.
8) Spin at 4º C for 10 minutes at 10,000 rpm in the Sorvall RC-5B, using the HB-6 rotor (16342g). While this is spinning, set the ultracentrifuge to cool. (Set to 70,000 rpm, 4°C, 1.5 hours).
9) The previous spin should separate the oocytes into 3 layers: lipid(top), cytoplasm(center), and yolk and pigment(bottom). Any excess Versilube from previous steps will appear as bubbles in the cytoplasmic layer. (The cytoplasmic layer should make up more than half of the volume in the centrifuge tubes.)
10) Remove the cytoplasmic layer (which should be grey to brown/gold-but may be darker or lighter depending on the pigment of the oocytes; in general this does tend to be darker in the oocyte prep than in the egg extract) with a drawn out disposable glass pipet. (pull under a flame and cut off the tip). A 3-5 mL syringe with a long, bent needle also works very well. Transfer to a 15 ml graduated tube on ice to measure volume. (You should be able to fill your 15 ml centrifuge tube at least 3/4.)
11) Add a 1:1000 dilution of the 10 mg/ml protease inhibitors (pepstatin, chymostatin & leupeptin) to the cytoplasm.
12) Spin at 4ºC for 10 minutes at 10,000 rpm using the HB-6 rotor. Collect the cytoplasmic layer as described in step 10 and transfer to ultracentrifuge tubes (Beckman 349622). Fill each at least halfway, but not high enough to lose volume during a fixed angle spin. Balance tubes carefully.
13) Spin in ultracentrifuge at 4ºC for 1.5 hours at 70K rpm, no brake (set deceleration to zero) in the Beckman TLA100.4 rotor. After this spin you will have 4 layers, top to bottom: lipid, cytosol, membranes/mitochondria and glycogen/ribosomes.
14) Remove the cytosolic layer, place in iced ultracentrifuge tube(s). Rebalance.
15) Spin at 4ºC for 20 minutes at 70K rpm, no brake. Remove cytosolic layer, transfer into eppendorf tubes on ice.
16) Aliquot 10-15mL into small eppendorf tubes and snap freeze in liquid nitrogen. Store in -80°C freezer.
Setting up a transgenic station
Dissecting microscope & light
Harvard Apparatus 11 Syringe Pump (single or dual syringe)
Hamilton Gas-tight Syringes, 25µl
Mineral Oil (Sigma M-8410)
Tygon Tubing, ID 1/32", OD 3/32", wall 1/32" (Fisher 14-169-1A)
micromanipulator (Narishige, etc.)
1) Fill a Hamilton gas-tight syringe (25µl) and a length of tygon tubing (long enough to reach from the pump to the micromanipulator) with mineral oil. Use the plastic rod that comes with the micromanipulator as your "needle holder". The injection needle filled with your transgenic reaction is directly inserted into the tygon tubing, and the needle is fixed to the manipulator rod by tightening the screw on the rod.
2) Set the pump to the desired speed (18.0µl/hr, diameter: 0.73 mm for 25µl Hamilton Gas-tight Syringe). Wait for the backpressure to subside, start the pump, and inject at a rate of approximately one egg per second. Remember to check periodically for bubbles in the tubing and clogs in the needle. The needle can be intermittently lifted out of the injection media to remove any debris that gathers at the tip.
Preparation of injection needles
Preparation of proper needles is essential for the success of the transgenic procedure. The nuclear transplantation needles should have a long sloping taper from the widest part of the needle to the tip (12-15 mm) to control the flow rate through the large needle tip. The tip must be large enough to allow sperm nuclei to pass through unharmed, but small enough to prevent large wounds in the egg (70-80µm). The tip should be beveled to allow piercing of the cell membrane with as small an amount of damage as possible.
NOTE: Previous protocols have suggested using a siliconizing agent to coat injection needles to prevent shearing of sperm nuclei. Our experimental evidence suggests that this process is not necessary.
Use glass capillaries (World Precision Instruments, TW100-4, OD/ID = 1.0/0.75 mm) for making injection needles. Pull several dishes worth of needles using a capillary puller (Sutter Instruments P-87; heat=900, pull=15, velocity=200, time=5).
To produce the correct tip size and shape, place a pulled capillary over a stage micrometer under a dissection microscope, and clip the tip to 70-80µm. Two suggested methods to produce the appropriate bevel shape in the needle tip are: A) a pair of forceps that have uneven ends can be placed parallel to the axis of the needle and the end of the needle broken to the correct size, or B) two breaks can be made using a normal pair of forceps—the first to set the diameter of the needle tip opening, the second to tap off one “side” of the tip to produce the bevel.
Preparation of agarose coated injection dishes
Nuclear transplantations are performed in a petri dish that has a square depression within an agarose bed. To prepare dishes, pour molten agarose (1% agarose in 0.4x MMR with Ca2+) into several 60-mm Petri dishes. Before the agarose solidifies, float a square plastic plate (2.5 x 2.5 cm cutouts of plastic pipet tip boxes work well) on the surface so that a depression will be formed after the agarose solidifies. Ensure that no bubbles are present under the plastic plate—remove and refloat if necessary. After the agarose solidifies, carefully remove the plastic plates, wrap the openings in Parafilm, and store at 4°C. A typical dish will hold 300-400 X. laevis eggs.
Preparation of special tips
·Cut-off p200 tips to handle sperm nuclei
The tip should be cut with a clean razor blade to approximately 1 mm inside diameter.
·Sperm nuclei loading tips
Attach 5 mm piece of Tygon Tubing (see below) to the tip of cut-off p200 tips.
Things to be prepared the day before the REMI transgenesis
Injection medium and 10% BSA
Thaw the -20°C stocks and store at 14°C
Culture medium (500 ml)
10 x MMR (with Ca2+) 15 ml (final 0.3x)
10% BSA 25 ml
distilled water 460 ml
50 mg/ml gentamycin 0.5 ml
pH to 7.5 with NaOH. Phenol Red may be added as a pH indicator (0.5 ml of 0.5 % solution (GIBCO-BRL, 15100-043)). Sterilize by filtration or use sterile water for the preparation. Store at 14°C.
Post-dejellying rinse solution (300 ml)
10 x MMR (no Ca2+) 30 ml (final 1.0x)
10% BSA 15 ml
distilled water 255 ml
Sterilize by filtration or use sterile water. Store at 14°C.
Prepare female frogs
Inject HCG (400µl of 2 units/µl solution) to a female the night before the sperm nuclear transplantation (use 1 ml syringe with 27G needle). Keep the injected female in a 16°C incubator for overnight. Usually the frog start laying eggs 11 hours after the injection.