Transgenesis in Xenopus laevis - adapted from Amaya et al 2001 (Conlon lab)

From Xenbase
Revision as of 15:17, 22 November 2011 by Anonymous (talk) (Created page with "Protocol submitted by VGP from Conlon lab protocols [http://www.unc.edu/~fconlon/Protocols.htm#tropprot] link to protocol document [http://www.unc.edu/~fconlon/Protocols/Transg...")
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)
Jump to: navigation, search

Protocol submitted by VGP from Conlon lab protocols [1]


link to protocol document [2]


Transgenesis in Xenopus laevis Adapted from that of Amaya et al. (21 January, 2001)


A. High Speed Egg Extract Preparation


Stock Solutions

To be made in advance of prep:


10X Marc’s Modified Ringers (MMR) For 1L

1M NaCl (Fisher S271-3) 58.44 g NaCl
20mM KCl (Fisher P217-500) 1.49 g KCl
10mM MgCl2·6H2O (Sigma M-9272) 2.033 g MgCl2·6H2O
20mM CaCl2·2H2O (Sigma C-5080) 2.94 g CaCl2·2H2O
50mM HEPES (Sigma H-3375) 11.92 g HEPES
dH2O dH2O to 1L
pH 7.5 pH to 7.5
  • Autoclave to sterilize and store at room temperature.


20X Extract Buffer Salts (XB Salts) For 1L

2M KCl (Fisher P217-500) 149.12 g KCl
20mM MgCl2·6H2O (Sigma M-9272) 4.066 g MgCl2·6H2O
2mM CaCl2·2H2O (Sigma C-5080) 0.294 g CaCl2·2H2O
  • Filter sterilize and store at 4oC.


1.5M Sucrose

51.35 g Sucrose (Mallinckrodt AR 8360)
dH2O to 100 mL
  • Filter sterilize and store in 8.33mL aliquots at -20oC.


1M HEPES

23.83 g HEPES (Sigma H-3375)
dH2O to 100 mL
  • Titrate with 10N KOH so that when diluted to 15mM, pH is 7.7 (This should require about 5.5mL 10N KOH for 100mL.)
  • Filter sterilize and store in 0.75mL aliquots at -20oC.


1M CaCl2 For 250mL

CaCl2·2H2O (Sigma C-5080) 36.75 g CaCl2
  • Filter sterilize and store at 4oC.


1M MgCl2 For 100mL

MgCl2·6H2O (Sigma M-9272) 20.33 g MgCl2·6H2O
  • Filter sterilize and store at room temperature.


200mM EGTA For 100mL

EGTA (Sigma E-3889) 7.61 g EGTA
PH 7.7 pH to 7.7 with NaOH
  • Autoclave to sterilize and store at room temperature.


Energy Mix For 5mL

150mM Creatine Phosphate (Roche 0621714) 0.2454 g Creatine Phosphate
20mM ATP (Roche 519979) 0.061 g ATP
20mM MgCl2·6H2O (Sigma M-9272) 0.020 g MgCl2·6H2O
  • Store at –20oC in 0.1mL aliquots.


10mg/mL Leupeptin For 1mL

Leupeptin (Roche 1017101) 0.010 g Leupeptin
dH2O dH2O to 1mL
  • Store at –20oC in 40l aliquots.


10mg/mL Chymostatin and Pepstatin For 1mL

Chymostatin (Roche 1004638) 0.010 g Chymostatin
Pepstatin (Roche 1359053) 0.010 g Pepstatin
dH2O dH2O to 1mL
  • Store at –20oC in 40l aliquots.

Versilube F-50 (General Electric) (Not necessary for protocol)


100U/mL Pregnant Mare Serum Gonadotropin (Calbiochem 367222)

  • Make up in sterile water and store at –20oC in 10mL aliquots.


1000U/mL Human Chorionic Gonadotropin (Sigma CG-10)

  • Make up in sterile water and store at 4oC.


+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++


B. Sperm Nuclei Preparation


Stock Solutions

To be made in advance of prep:

10X Marc’s Modified Ringers (MMR) For 1L

1M NaCl (Fisher S271-3) 58.44g NaCl
20mM KCl (Fisher P217-500) 1.49g KCl
10mM MgCl2·6H2O (Sigma M-9272) 2.033g MgCl2·6H2O
20mM CaCl2 (Sigma C-5080) 2.94g CaCl2
50mM HEPES (Sigma H-3375) 11.92g HEPES
dH2O dH2O to 1L
pH 7.5 pH to 7.5
  • Autoclave to sterilize and store at room temperature.


1.5M Sucrose

51.35g Sucrose (Mallinckrodt AR 8360)
dH2O to 100mL
  • Filter sterilize and store in 8.33mL aliquots at -20oC.


1M HEPES

23.83g HEPES (Sigma H-3375)
dH2O to 100mL
  • Titrate with 10N KOH so that when diluted to 15mM, pH is 7.7 (This should require about 5.5mL 10N KOH for 100mL.)
  • Filter sterilize and store in 0.75mL aliquots at -20oC.


10mM Spermidine Trihydrochloride

0.128g Spermidine Trihydrochloride (Sigma, S-2501, store at -20oC)
50mL dH2O
  • Filter sterilize and store in 2mL and .5mL aliquots at -20oC.


10mM Spermine Tetrahydrochloride

0.174g Spermine Tetrahydrochloride (Sigma, S-1141, store at -20oC)
50mL dH2O
  • Filter sterilize and store in 1mL aliquots at -20oC.


0.1M KCl

0.746g KCl (Fisher P217-500)
dH2O to 100mL


100mM Dithiothreitol (DTT)

0.154g DTT (Roche 100-032)
dH2O to 10mL
  • Filter sterilize and store in 0.5mL aliquots at -20oC.


500mM EDTA

18.61g EDTA (Sigma E-5134)
dH2O to 100mL
  • Store at room temperature.


100% Glycerol (Fisher G33-500)

  • Autoclave to sterilize and store at room temperature.


10mg/mL Hoechst Stain

10mg Hoechst Stain (Sigma B-2261, No. 33342)
dH2O to 1mL
  • Divide into 0.5mL aliquots, wrap tubes in foil and store at -20oC.


Sperm Dilution Buffer (SDB)

250mM Sucrose 3.3mL 1.5M Sucrose
75mM KCl 15mL 0.1M KCl
0.5mM Spermidine Trihydrochloride - 1mL 10mM S. Trihydrochloride

0:.2mM Spermine Tetrahydrochloride - 0.4mL 10mM S. Tetrahydrochloride

0.3mL dH2O
  • Add about 80l 0.1N NaOH per 20mL solution to titrate to pH 7.3-7.5.
  • Store in 1.0mL aliquots at -20oC.


+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++


The night before prep:

1. Inject 2 males with 500U HCG. (This can also be done very early – 6am – on the day of the prep if the prep will not be completed until late afternoon.)


On the day of prep:

1. Prepare the following solutions:

1X MMR

45mL H2O
5mL 10X MMR


2X Nuclear Preparation Buffer (NPB)

500mM Sucrose 8.33mL 1.5M Sucrose
30mM HEPES 0.75mL 1M HEPES
1mM Spermidine Trihydrochloride - 2.5mL 10mM Spermidine Trihydrochloride
0.4mM Spermine Tetrahydrochloride - 1mL 10mM Spermine Tetrahydrochloride
2mM DTT - 0.5mL 100mM DTT
2mM EDTA - 0.1mL 500mM EDTA
11.8mL H2O
  • Store on ice.


1X NPB

10mL H2O
10mL 2X NPB
  • Store on ice


0.1% Aminobenzoic Acid Ethyl Ester (Tricaine)

1.0g Tricane (Sigma A-5040)
1.0g Sodium bicarbonate (0.1%) (Fisher S233-500)
H2O to 1L

2. Anesthetize 2 male Xenopus laevis frogs in 0.1% Tricaine for at least 20min.

3. Using fine forceps and dissecting scissors, remove the two testes from the frog and fat bodies while being careful not to puncture the testis.

4. Remove as much fat and blood as possible. It is useful to roll the testes along a clean paper towel as the fat and blood will stick to the towel.

5. Place the testis in a 35mm tissue culture dish containing cold 1X MMR.

6. Rinse the testis in 3 changes of cold 1X MMR.

7. Place the cleaned testes into another 35mm dish with 5mL of cold 1X NPB for 2-5min.

8. Transfer the testes to a clean 35mm dish and macerate the tissue using clean forceps. No clumps should be visible to the naked eye.

9. Line a small funnel with about 4 thicknesses of cheesecloth. [This can be done by stacking 2 squares (about 8in.) of cheesecloth, folding them lengthwise, then widthwise, and opening them into a cone. Place the cone into the funnel.]

10. Place the funnel into a round bottom, 15mL tube (Falcon 2059).

11. Add 3mL cold 1X NPB to the culture dish and testes with a 10mL disposable pipet. Gently pipet the mixture up and down about 3X. Add the solution to the center of the cheesecloth.

12. Rinse the culture dish with 2mL cold 1X NPB and add to the cheesecloth.

13. Add an additional 5mL cold 1X NPB to the cheesecloth directly.

14. With a gloved hand, gently squeeze the remaining liquid from the cheesecloth through the funnel and into the tube. The final volume of suspension in the tube should be about 9mL.

15. Spin the suspension 10min. at 3,000rpm at 4oC. (Sorval RC5C Centrifuge Model, Sorval HB-4 rotor with appropriate adaptors or Beckman J13 rotor)

16. During the spin:

a. Place 1mL 1X NPB at room temperature to equilibrate.
b. Make up a 10% BSA solution

1g BSA

dH2O to 10mL
*Add water to 15mL tube first, then add 1g BSA, place on rocker to get into solution.

17. Take the sperm from the centrifuge and pour off the liquid from the pellet (note: there will be some blood nuclei contamination at this point).

18. Resuspend the pellet in 9mL cold 1X NPB.

19. Spin the suspension 10min. at 3,000rpm at 4oC.

20. During the spin:

a. Dissolve 1mg L--lysophosphatidylcholine (Lysolecithin, Sigma L-4129) in 100l H2O at room temperature (It is easier to measure close to 1mg and add water accordingly). Be sure that the lysolecithin powder is completely dry before use (ie. not clumpy). If it is wet, discard the entire stock. Store the powder at -20oC wrapped in parafilm.
b. Make up 10mL 1X NPB with 3% BSA
2mL H2O
5mL 2X NPB
3mL 10% BSA
*Store on ice

21. Take the sperm from the centrifuge and pour off the liquid from the pellet (note: there will still be some blood nuclei contamination remaining).

22. Resuspend the pellet in the 1mL of room temperature 1X NPB with a disposable 10mL pipet.

23. Add 50l of the lysolecithin solution from 20a. and mix gently. Incubate at room temperature for 5min.

24. Add 10mL of cold 1X NPB + 3% BSA, mix gently (but very thoroughly!).

25. Spin the suspension 10min. at 3,000rpm at 4oC.

26. During the spin:

a. Make up 1X NPB + 0.3% BSA
2.35mL H2O
2.5mL 2X NPB
0.15mL 10% BSA
*Store on ice
b. Make up Sperm Storage Buffer
85l H2O
250l 2X NPB
15l 10% BSA
150l 100% glycerol

27. Pour off the liquid from the pellet. The pellet should now be wider and loose, and should no longer contain any red areas.

28. Resuspend the pellet in 5mL 1X NPB + 0.3% BSA and spin the suspension 10min. at 3,000rpm at 4oC.

29. Carefully pour off the liquid and resuspend (using a blue tip with the end cut off) the sperm in 500l (2 frogs) sperm storage buffer. Transfer the suspension to a 1.5mL eppendorf tube and store at 4oC for up to 48hrs before counting, aliquotting and freezing.

30. To count the nuclei, cut the end off of a yellow tip and dilute 1l of the sperm suspension with 99l sperm dilution buffer (1:100).

Add 1l of a 1:100 dilution of Hoechst stock.

Transfer the solution to a haemocytometer for counting and visualize the sperm under a fluorescence microscope with a DAPI/Hoechst filter set.

A 1:100 dilution should result in 125-200x104 nuclei/mL (125-200 nuclei per large grid).

If greater, dilute accordingly with sperm storage buffer.

If substantially lower, repellet the nuclei at low speed (or let settle for a few hours) and resuspend in smaller volume.

31. Aliquot sperm nuclei final solution into 25l aliquots in 0.2l PCR tubes. Freeze in liquid nitrogen and store at -80oC. One aliquot can then be thawed on ice for each day of transgenesis.


C. Preparation of Needles, DNA and Equipment

  • These steps should be carried out the day before transgenesis as to avoid rushing.

1. Preparing nuclear transplantation needles.

Needles: 30l Drummond micropipets (Fisher 21-170)
Puller: Sutter Instruments Co. Flaming/Brown Micropipette Puller Model P-87
-The setting depends on the filament used, but should be adjusted so that a gentle slope in the needle is achieved (ex. p=50, v=100, t=5).
-Once needles have been pulled, the needles must be clipped to a diameter of 80-100m (40-60m for X. tropicalis). To do this, place the needle with the tip hanging over the edge of a flat box/dish under the dissecting scope. Set the magnification to 2.5x (total 25x) and align the needle with the ocular micrometer/graticule. Using fine forceps (an uneven pair works well for this) gently clip the needle so that the tip diameter is approximately 3-4units on the graticule and beveled.

2. Preparing agarose coated dishes.

-Fill 60mm tissue culture dishes (Falcon Easy Grip 60x15mm, 35-1016) to the center line with 1% agarose (electrophoresis grade) in 0.1X MMR.
-Place a small weigh boat into the agarose to create a square depression in the cooled agarose. Place the inverted dish lid onto the weigh boat to hold it in place until the agarose cools.
-Wrap the dishes in parafilm and store at 4oC.

3. Preparing linearized DNA.

-Digest the plasmid DNA using standard conditions, usually overnight. It is useful to digest 3g of DNA in a 30l reaction to achieve a final concentration of 100ng/l as this is the concentration needed for future steps.
-Check the DNA on a gel to assure digestion.

4. Injecting apparatus.

-Pump: Harvard 22 Basic Syringe Pump, Harvard Apparatus #55-2222
X. tropicalis = rate-0.200l/min., diameter-1.46mm, syringe-0.1mL
X. laevis = rate-0.600l/min., diameter-7.28mm, syringe-2.5mL
Keys = To set rate, press rate + set simultaneously
To rotate through rate settings, press rate + set, then press rate
To fill mineral oil, attach a 3ft. piece of Tygon tubing to a wide gauge needle, fill the syringe with mineral oil and remove any bubbles, attach the syringe to the needle, mount the syringe in the injector pump and tighten, set the rate to ~999 and start to clear the air bubbles from the tubing, reset the pump
-Syringes: Hamilton GasTight
X. tropicalis – 0.1mL #1710, Fisher #SZR-635-010Y
X. laevis – 2.5mL #1002, Fisher #SZR-635-040W
-Needles: Drummond 30l Micropipettes, SLS 1-00-0300 or Fisher 21-170
-Tubing: Tygon 0.8mm x 0.8mm R3603 Tubing, Fisher 14-169-1A
    • Diameter is key!!
-Oil: Embryo tested mineral oil, Sigma M-8410
-Microscope: Leica MZ-6
-Ocular micrometer: 5mm reticule for widefield 10X/21B, Leica 394771


D. Transgenesis by Sperm Nuclear Transplantation

To be made in advance:

1X MMR + 6% Ficoll + Gentamycin

20mL 10X MMR
180mL dH2O
12g Ficoll (F-4375)
200L Gentamycin (50mg/mL in H2O, stored at -20oC) (Sigma G-4793) (final 50g/mL)
  • Filter sterilize, store at 16oC (Corning Incorporated 500mL Bottle Top Filter with 45mm neck, 0.22m Cellulose Acetate, #430513)

0.1X MMR + 6% Ficoll + Gentamycin

5mL 10X MMR
455mL dH2O
30g Ficoll
500L Gentamycin (50mg/mL in H2O, stored at -20oC)(final 50g/mL)
  • Filter sterilize, store at 16oC

100mM MgCl2

0.102g MgCl2
dH2O to 5mL
  • Store at 4oC in 0.25mL aliquots if making in advance

The night before injections will be completed:

1. Inject 2 female Xenopus laevis individuals with 500U (usually in 0.5mL sterile water) human chorionic gonadotropin into the dorsal lymph sac. Place the injected females in a 19oC incubator for 10-12hrs.

To be made on the day of injections:

1X MMR

200mL 10X MMR
1800mL dH2O
  • Easiest to make up in a large graduated cylinder to be used for the day

2.5% Cysteine

7.5g L-cysteine hydrochloride 1-hydrate (L-cysteine for X. tropicalis)
225mL 1X MMR
pH to 7.8-8.0 with 10N NaOH
1X MMR to 300mL
  • Make up in a 600mL beaker

0.4X MMR + 6% Ficoll + Gentamycin

35mL 0.1X MMR + 6% Ficoll + Gentamicin
15mL 1X MMR + 6% Ficoll + Gentamicin
  • Make up in a 50mL disposable tube

Before beginning, gather the following materials in addition to the above solutions:

-1 tube sperm nuclei from -80oC, place on ice
-1 tube egg extract from -80oC, place on ice
-1 tube sperm dilution buffer from -20oC, place at room temperature
-Linearized DNA constructs (100ng/l), place on ice
-1cm pieces of 0.8x0.8mm silicon tubing (Tygon R3603), one per construct
-Cut off p200 tips
-Razor blade (for cutting tips)
-2x2 square of parafilm (for cutting tips)
-4-1.5mL eppendorf tubes
-Restriction enzyme (Not I or Sal I are ideal as long as they don’t cut the linear DNA)
-2 agarose-coated dishes per construct
-Pulled and clipped needles
-2 clear 2” tall, flat top boxes filled with ice (to place injecting dishes on under the microscopes)
  • Complete procedure for one construct at a time. (It is also possible to do this for 2 constructs once proficient with one.)

1. Label the four eppendorf tubes 1, 2, 3, 4.

2. Add the following into tube #1 and leave at RT for 5min:

1l Linearized DNA construct
5l Sperm nuclei **USING CUT OFF p200 TIP**

3. In the meantime, cut off 3 long, glass pipets to create a wider tip and flame the ends to create smooth edges. These will be used to transfer eggs and embryos.

4. Also during the incubation, add the following to tube #2:

19l Sperm dilution buffer
2l 100mM MgCl2
2l Egg extract **USING CUT OFF TIP**

4. With approximately 30sec. left on the incubation, add the following into tube #3:

9l dH2O
1l Restriction enzyme (Not I or Sal I)
*tap tube to mix

5. After the 5min. incubation, add 1l of the diluted enzyme to tube #2.

6. Use a cut off p200 tip to transfer the contents of tube #2 into tube #1 and GENTLY pipet to mix. Do not create any bubbles in the solution (don’t completely empty the tip until the very end).

7. Incubate at room temperature for 15min.

8. During the incubation, squeeze two females into 2 medium beakers to release their eggs (squeezing into separate beakers controls for contamination will poor quality eggs).

9. Immediately add approximately 100mL-200mL 2.5% cysteine in 1X MMR. Leave eggs for about 5min. or until jelly coat is removed completely and eggs pack tightly into the bottom of the beaker. Only swirl gently if absolutely necessary.

10. Pour off most of the cysteine and rinse the eggs 4-5 times with fresh 1X MMR.

11. Fill 2 agarose coated dishes with 0.4X MMR + 6% ficoll + gentamicin.

12. With a cut off glass pipet, transfer the eggs to the two agarose coated dishes and place on the ice-filled box under the dissecting scopes.

13. Add 150l of sperm dilution buffer to tube #4.

14. After the 15min. incubation is finished, carefully transfer 5l of the reaction mixture USING A CUT OFF TIP into tube #4.

15. Place a piece of Tygon tubing onto the end of a cut off p200 tip and use this to very gently mix the reaction.

16. With the p200 set at 150, fill the Tygon tubing and the tip until the plunger of the pipet is half way in. With your thumb remaining on the plunger, carefully remove the tip from the p200 and hold it horizontally.

17. Insert the back end of a needle into the tubing and place vertically until the needle is filled. Once filled, remove the tubing from the needle.

18. Insert the back end of the needle into the Tygon tubing attached to the Harvard apparatus and mount in the injecting stand.

19. With the correct settings, inject the eggs with a quick, fluid motion.


E. Culturing the Embryos

1. Leave the injected embryos in the agarose coated dishes and place them in a 16oC incubator.

2. When the embryos reach the 4 cell stage (about 3 hours), transfer the correctly cleaving embryos using a cut off glass pipet (flamed to remove sharp edge) into 10cm Petri dishes with 0.1X MMR + 6% Ficoll +Gentamycin.

3. Place the embryos back into the incubator and periodically remove any dead embryos.

4. Once the embryos have completed gastrulation (the following afternoon), transfer them to 0.1X MMR + Gentamycin (no Ficoll) and return them to the incubator or leave at room temperature.

5. Periodically remove any dead embryos from the dish.

6. Visualize and photograph any GFP constructs using UV light and the appropriate microscope filter.


F. Other Important Information

1. Amaya Lab members performing transgenesis:

Enrique Amaya, ea3@mole.bio.cam.ac.uk
Shoko Ishibashi, si226@cam.ac.uk
Roz Friday, rf231@cam.ac.uk
Jeff Huang, jh424@cam.ac.uk

2. Instead of GFP, an alkaline phosphatase construct and protocol are available from Jeff Huang (jh424@cam.ac.uk). This would allow for the visualization of earlier expression.