Xenopus oocytes and embryos: *

(D.R. Smith)

At least the day prior to the micro-injection prepare the following:

Pull glass micro-injection needles with a micro-electrode puller. Under a binocular microscope break the end of the needle with a tapered tungsten filament (produced by vaporizing the end of a tungsten wire with an oxy-acetylene torch) fused into the end of glass tube as holder. The end of the needle is broken to a diameter of 10-20 um. The tungsten filament is simply used to apply force to the needle. Compare the broken needle end with a standard needle under a magnification of 30x or more and rebreak if necessary. Select needles with single near 45^O angled single points. (It is useful though not essential to mark the point side of needle. The point is then oriented to be on the lower needle surface when injecting.

Prepare small discs of nylon netting of the correct size to fit 9cm petri dishes. Pipette about 1 ml of chloroform into the bottom of the petri dish. Leave for between 10 and 30 seconds. Remove chloroform, and place one of the discs into the bottom of the dish. Press net firmly down against petri, moving around the edge of the netting. When the chloroform appears dry the netting disc should be firmly and evenly stuck to the bottom of the disc. To aid evaporation of chloroform while fixing netting in place a hair dryer can be used or the petri simply held in the airflow into the fume hood. Sterilise dishes with diethyl pyrocarbonate (0.1% in H2O made up freshly) for about 30 mins then wash three to four times with sterile distilled water. Store closed until required. UV sterilisation under the lamp of a culture hood is often also performed just before use.

DNA or RNA solutions to be injected should be clean (if possible have been purified on a caesium chloride gradient without EtBr) and the concentration should be accurately known. The DNA should be dissolved in injection buffer (10 mM Tris HCl pH 7.5, 88 mM NaCl). From this make up a 10x injection stock. The actual concentration of this 10x stock will vary depending upon what is to be injected. For the standard polymerase I vector (pX1108c) the concentration is 250 ng/ul of insert. For the M13 ribosomal promoter insert the concentration is 280 ng/ul. From the 10x stock, dilute down to a 1x micro-injection stock, still in injection buffer. This can then be used directly in the injection mix (if doing a competition or internal standard type experiment) as follows:

Mix the solutions well and spin briefly. Store at -20oC until required and then on ice.

Make up stock solutions and autoclave. For 5 litres of 1x Barths media, take 4 litres distilled H2O, add 88 ml 5 M NaCl and 50 ml of the three stock solutions. Adjust to pH 7.6 with conc HCl. Make up to 5 litres dispense into 500 ml bottles and autoclave. Store at 4oC until just prior to use, then transfer to 18oC incubator.

Aliquot 2 ml from the stock bottle (100x pen. strep. (Gibco)) into 4 ml tubes. Store at -20oC. Thaw and use as required. Add one 5 ml aliquot to each 500 ml bottle of Barths media immediately prior to use.

Sterilise all the operating equipment that will be required, such as scissors, tweezers and arterial clamp. Wash the equipment thoroughly, rinse with distilled water then bake in an oven at 180oC for 2 hours or autoclave.

(These have now been superceded by 1ml pipette tips cut to the appropriate size just before use and flamed). Free oocytes are manipulated using a pasteur pipette whose tip has been broken off to yield a wide lumen and flamed. Sterilise by baking at 180oC for 2 hours.

Oocytes are taken from Xenopus laevis or Xenopus borealis females. Best results are obtained from females which have been aclimatised to the lab for several weeks and a week or more after an injection of 50 to 100u of hCG. For removal of the oocytes, the frog is narcotised in 0.5 litre of MS 222 (Sandoz or Sigma) at a concentration of 1 to 1.6 g/litre in tap water at near room temperature. After 5 to 12 minutes exposure (2 min after stimulated movement ceases), depending on size, the frog remains anaesthesized for 1/2 to 1 hour. The frog is placed on its back, on a wet cloth and rinsed with Barths plus antibiotic berfore and at regular intervals during operation. An incision about 1 cm long, slightly offset from the ventral line, is made in the skin. The peritoneum and the muscle tissue are cut open. A small portion of the ovary is pulled out with forceps and removed with a pair of scissors. The clump of oocytes is immediately transferred to a petri dish containing modified Barth medium with antibiotic. The rest of the ovary is pushed back into the body cavity. The peritoneum and the muscle tissue are sewn up and then the skin closed off using cat gut. The oocytes are kept at +18oC or at 4oC and can be used for at least 2 days.

Oocytes of stage 5 and 6 are separated from the ovary with a platinum wire loop. These can be distinguished morphologically from the other developmental stages by a change in the animal pole from a dark brown to a light brown or beige, and the formation of a rather distinct lighter coloured boundary between the hemispheres. This colour change occurs when the oocyte reaches a diameter of 1000 to 1200 um. Stage 5 represents about 10% of the stage 2 to stage 6 population. Stage 6 oocytes are identified by their relatively unpigmented equatorial band, which measures about 0.2 mm wide. The animal hemisphere returns to a dark brown colour similar to that of the earlier stages. The size of the oocytes is 1200 - 1300 um and polarisation of yolk platelets in the vegetal hemisphere and smaller platelets in the animal hemisphere is readily apparent.

Free oocytes are manipulated using a pipette tip with a wide lumen. About 40 oocytes are manipulated on to the petri dishes containing the bonded netting. The oocytes are orientated on the netting in such a way that the brown animal pole is upwards. The petri dish and its contents are placed in an MSE 6L or Sorvall RC3, 6x 1 litre rotor bucket and centrifuged for about 10 mins at 18oC at 1000-1600 rpm. The results of centrifugation are variable and it is often best to carry out a small series of tests to determine the best centrifuge conditions. Centrifugation is considered optimal when the dark pigment ring is just displaced, indicating the position of the nucleus. During the centrifugation the oocytes are forced into the meshes of the nylon netting and remain fixed. When the centrifuge has come to a stand still the oocytes will present themselves with the brown pigment ring facing the injector. Centrifuged ocytes are best used immediately. Centrifugation of the oocyte does not impair the capacity to synthesize RNA.

When using the positive displacement system, the needle is filled with paraffin oil from its non-tapered end by means of a syringe. Care must be taken to ensure that no air bubbles are introduced into the needle. The needle is fixed to the micro-manipulator via the needle holder and a short length of PVC tubing, also filled with paraffin oil, again being careful not to introduce air bubbles.

1.6 to 3 ul of 1x injection solution is delivered from a standard tip onto a piece of Parafilm. From now on all operations are carried out under the binocular microscope. The injection needle is introduced into the drop with the aid of the micro-manipulator. The solution is aspirated into the needle by turning the micrometer screw slowly, to avoid shearing the DNA. The hydraulic system must be free of air bubbles so that when suction is applied, the miniscus within the needle rises immediately and stops rising as soon as the the micrometer stops turning. When most of the liquid has been sucked up, apply positive pressure until a small volume of solution is ejected. Then remove the needle from the remaining drop of liquid and immediately immerse the needle in some buffer to prevent drying until required.

Place the petri dish containing the centrifuged oocytes under the binocular microscope. The needle attached to the micromanipulator is aimed at the oocytes at an angle of about 60-70o. The oocytes are aligned relative to the tip of the needle by manoeuvering the petri dish. Before injecting the first oocyte it is advisable to ensure that the system is working by pressing the foot control until a drop of liquid can be seen. Repeat at least once through the course of the injection (i.e. after about 20 oocytes). The tip of the needle is introduced into the dead center of the ring of displaced pigment and just below the surface of the oocyte. 20 nl of the injection fluid is injected into each oocyte nucleus using the micrometer movement. An alternative method is to continuously run the drive motor running and to count the seconds that the needle remains in the oocyte. This method has the great advantage that it is extremely reproducable and that there is less chance that the needle will become plugged by yolk. This method is also less sensitive to any air within the hydraulic system.

Any uninjected oocytes are removed from the nylon with a jet of media. The oocytes are then incubated at 18oC (23oC must not be exceeded) for the desired time (about 14-18 hours).

Injected and incubated oocytes are removed from the nylon mesh by a jet of media as described above, and oocytes showing an unequal distribution of pigment are rejected. The remaining oocytes are transferred to a disposable 4 ml or eppendorf tube. Remove all of the barths media and resuspend in 400ul room temperature TE or RSB:

10 mM Tris HCl pH 7.5	200 ul 1 M
1.5 mM MgCl2	30 ul 1 M
10 mM NaCl	40 ul 5 M
	________
total volume	20 ml

Add 50 ul Proteinase K (20 mg/ml), gently whirlimix and leave 1 min. Add 50 ul 20% SDS and whirlimix vigorously until all the oocytes have lysed. Incubate for about 1 hour at room temperature.

After the above stage the oocytes can either be frozen or they can be taken through the next steps.

Add 50 ul 3 M NaAc (or NaCl) and whirlimix. Extract 3 times with phenol-chloroform and once with chloroform.

Add 2.5 vol. ice cold EtOH, mix well and leave at -20oC 1hr.

Spin 12,000 rpm, 10 mins and 4oC in Sorvall RC-5B, pour off supernatant and wash with 80% EtOH 12,000 rpm, 5 mins.

Dry samples (but be careful not to over dry) and for S1 mapping resuspend in formamide at a concentration of 4 ul/oocyte. Resuspension can prove to be difficult. Incubation at 65oC for a short time can help.

1. Glass capillary needles (broken)

2. Petri dishes (with netting) and a few without netting

3. Stock solutions of DNA

4. Modified Barths media

5. Antibiotic solution

6. Operating equipment (sterilized by baking)

7. Optional wide lumen pipettes (sterilized by baking) or 1 ml cut pipette tips lightly flamed.

8. Operate frog to recover ovary fragment

9. Remove oocytes with wire loop

10. Manipulate about 40 oocytes onto a tray (for each solution to be injected

11. Start centrifugation of one tray of oocytes (10 mins, 1,200 to 1,600 rpm, 18oC)

12. Fill needle with paraffin oil from blunt end using syringe

13. Insert into needle holder

14. Put 3 ul of injection solution onto piece of parafilm

15. Under binocular microscope slowly aspirate solution

16. Place tip of needle in buffer until ready to inject

17. Under binocular microscope inject each oocyte

18. Remove any uninjected oocytes with a jet of medium

19. Incubate at 18oC 14-18 hours

20. Resuspend in 500 ul RSB

21. Add 50 ul Proteinase K (20 mg/ml), mix and leave 1 min.

22. Add 50 ul 20% SDS, whirlimix until oocytes totally lysed

23. Incubate at room temp 1 hour

24. Add 50 ul 3 M NaAc (or NaCl).

25. Extract 3 times with 1 volume phenol/chloroform

26. Extract once with 1 volume chloroform

27. Add 2 volumes ice cold EtOH, mix well, -20oC 1hr

28. Spin 12,000 rpm, 10 mins and 4oC. Pour off S/N and discard

29. Wash with 80% EtOH 12,000 rpm, 5 mins and 4oC. Pour off S/N and discard

30. Dry pellet lightly

31. Redissolve in formamide at a concentration of 4ul/oocyte

32. Store at -20oC or -70oC.

Female Xenopus are induced to ovulate with human Chorionic Gonadotropin (CG) from ICN dissolved at 1000 I.U./ml in sterile distilled water or 1/10 Ringer's:-

1x Ringer’s:-

NaCl,	6.6gm
CaCl2,	0.15 gm
KCl,	0.15gm
NaHCO3	0.15 gm
	______
final volume	1 l

Normally females are injected with 100 I.U., 0.1ml, 2 days before eggs are required, then again with 400 - 700 I.U., 0.4-0.7 ml, 8 to 12 hr before.

Females may be kept in normal aquarium water or if the eggs they lay may be required for fertilisation, in High Salt-MBS.

Just before laying a male is sacrificed and the testes taken into 1 x MMR, 10% foetal calf serum, 100ugm penicillin, 50ugm streptomycin.

1x MMR,	1l:-
0.1M NaCl	20ml 5M NaCl
2mM KCl	1ml 1M KCl
1mM MgSO4	1ml 1M MgSO4
2mM CaCl2	2ml 1M CaCl2
5mM Hepes	10ml 0.5M Hepes
0.1mM EDTA	0.2ml 0.5M EDTA
Final pH 7.8.

Eggs are massage from female into 10 - 15 ml of 1 x MMR for immediate use or into High Salt-MBS for storage, (up to 12hr is claimed for very good batches, but this does not usually work for us). The testes is opened slightly at one end with forceps and wiped over the eggs in a minimal solution volume. The eggs are then flooded with 0.1 or 0.2 x MMR.

10x MBS (less CaCl2)

NaCl	51.3g
KCl	0.75g
MgSO4.7H2O	2.0g
HEPES	23.8g
NaHCO3	2.0g
pH to 7.6 with NaOH
Make up to 1l and filter sterilise.

MBS

10 x MBS (No CaCl2)	100.0ml
0.1M CaCl2	7.0ml
H2O	892.0ml
Gentamycin (50mg/ml)	1.0ml
(or 100u/ml penicillin, 100ug/ml streptomycin SO4 (Gibco))

High Salt MBS

10 x MBS (No CaCl2)	100.0ml
0.1M CaCl2	7.0ml
5M NaCl	4.0ml
H2O	888.0ml
Gentamycin (50mg/ml)	1.0ml

The first division occurs in about 1.5hr, the second after another hour. At the earliest 20 to 30 min. after fertilisation the eggs are dejellied in cysteine soln for two minutes, use 15 - 20 ml per batch of 50 - 100 embryos change after 2 to 3 min. for fresh if necessary. Keep time of dejelling as short as possible, around 5 min. though 6 to 10 min. has been used quite successfully as long as subsequent wash is thorough.

Cysteine solution, make fresh :- 100ml

cysteine HCl	2 gm
NaCl (5M)	1.6ml
10M NaOH to pH 7.8-7.9.	1.5 ml

Wash very thoroughly with 0.1 or 0.2 x MMR (e.g. 6 x 100ml) finally transfering into the injection buffer, 0.2 x MMR, 5% Ficoll 400.

Inject between 35 - 100 pgm of DNA or up to 2 ngm RNA in a volume as small as possible. Generally a maximum of 10 to 20 nl is used but smaller is better, much smaller volumes ~50pl have been used by some. Inject into the animal pole of one or two cell embryos.

Leave at 14 to 18oC in 0.2 x MMR, 5% ficoll 400 for a few divisions or up to 32 cell stage, then transfer to 0.1 x MMR and incubate O/N at 14 to 18oC adding 1ug/ml Gentamycin or penicillin streptomycin (100x solution Gibco) if necessary. Lower temperature incubation leaves embryos at gastrula next morning, but we have found that viability is often poorer than at higher temperature.

Embryos are placed in 0.1mM BrdU, 0.1 x MMR for 2 to 4 hr before fixation or further treament.

Islam & Moss (1996) TIGs 12, 459

Incubations were performed in a 24 well tissue culture plate and embryos contained in ~1ml plastic cups with nylon net bases. These were prepared from 1.5ml microcentifuge tubes by removing the tapered section of each tube and welding this to a fine nylon net. R/T incubations were performed on a Nutator, while those at elevated temperatures were performed in a shaking water bath.

The embryos are fixed at R/T in MEMFA (0.1M MOPS, pH 7.4, 1mM EGTA, 2mM MgSO4 and 3.7% formaldehyde for two or more hours, (possibly O/N). After fixation the embryos are transfered to methanol and can be kept many months at -20oC.

MEMFA; (0.1M MOPS, pH 7.4, 1mM EGTA, 2mM MgSO4 and 3.7% formaldehyde

The embryos are rehydrated at R/T by sequential washings in 75% methanol-25% PBST, 50% methanol-50% PBST, 25% methanol-75% PBST and finally PBST, each 5 min.

PBS; 0.145M NaCl, 10mM Na-phosphate pH 7.4

PBST; PBS containing 0.1% Tween 20)

The embryos are refixed in 4% paraformaldehyde in PBS for 20 min. at R/T.

Embryos are briefly rinsed in hybridising solution and then incubated in fresh hybridising solution (HB) at 75oC for 30 min.

Hybridising solution (HB); 5 x SSC pH6.0, 50% formamide, 50ug.ml-1 heparin, 0.1% Tween 20, 0.5mg.ml-1 torula RNA.

Without change of solution, simply lower the temperature to hybridising temperature (55 to 70oC) and incubate 4 to 5 hours.

Change HB for fresh preheated solution 30 min before beginning hybridisation. Add 500ng.ml-1 of digoxygenin labelled riboprobe and hybridise O/N.

Incubate at temperature of hybridisation Thyb in;

The embryos are brought to R/T and washed in PBST for 3 times, 5 min each.

The embryos were washed 12 x in 2 ml per well of PBST, 15 min each. The washings may be continued O/N at 4 oC if necessary.

Just prior to staining the embryos were washed in 2 changes of NTMT, 15 min each at R/T. They are then transfered to a clean 24 well plate and incubated at R/T in developing solution. Development is judged visually and the developing solution changed for fresh as soon as it becomes slightly coloured. Development is terminated by washing once or more with PBS and then fixed in MEMFA (0.1M MOPS, pH7.4, 2mM MgSO4, 1mM EGTA and 3.7% formaldehyde) for 5 min or more, (they may be stored in this solution at 4 oC).

The embryos may be cleared (in glass vials) by washing;

1 x PBS, 1x methanol, 1 x 50%, methanol-50% benzyl benzoate and finally in 1 part benzyl alcohol, 2 parts benzyl benzoate. This solution will leach some colour and so should not be used until just before photographic recordings are made.

After fixation as for wholemount or after wholemount hybridisation the embryos are embedded as follows:-

Either dehydrate stepwise, 10 min. each through;

or ;

Then 30 min. in each of;

The blocks are left at R/T to set then carefully placed at 4oC O/N. They are then sliced at 10um. 5 to six slices are cut from ribbon and dropped onto the surface of 45oC preboiled water in a constant temperature bath. The sections are then lifted out on a Tespa treated slide, see below. The slides are placed on a slide warmer at 37oC for 30 min. or just left standing to drain and finally placed in a slide box containg dessicant, sealed and heated at 37oC O/N. They are then stored in the same box with dessicant at 4oC until required.

8. Store at R/T dust free.

6. Store at R/T dust free.

4. Rinse in acetone for 10 s, then rinse in H2O for 10 s and bake at 160oC

To remove paraffin wax and rehydrate sections place slides for 5 min. in each of the following;

Originally for Zebrafish embryos. Strähle et al. TIG 10, 75-76, (1994).

Originates from Drechsel et al. Curr. Biol.7, 12-23.

TBSN:- 50mM Tris-HCl, 155mM NaCl, 0.1% NP-40.

PIPES	24g ,
EGTA	1.9g
MgCl2	1ml 1M
	-----------
final volume	200ml.

paraformaldehyde (Dissolve in PEMFA & water by boiling.)	4g
50% glutaraldehyde	1ml
10% Triton	2ml
5x stock PEMFA	20ml
	_____
Final volume	100ml

Originates from Drechsel et al. Curr. Biol.7, 12-23.

Lectins are present on the surface membrane of the embryo animal pole. By binding these with biotinylated soybean agglutinin before the first division, the recruitment of new and hence unbound membrane can be followed by subsequent staining with fluorescent or enzyme conjugated anti-biotin antibodies.

100 x Hoechst stock solution (20ug/ml in H2O, kept at 4oC) is diluted in H2O and the rehydrated slides treated with this for 5 to 10 min. Finally slides are washed in H2O and mounted in aqueous solution. The cover slips are sealed with clear nail varnish.