We have been using the transgenic procedure developed by Kroll and Amaya to produce transgenic Xenopus laevis. We encountered many problems with the procedure over the course of our experiments. Many of the problems (and solutions) are addressed by Kroll and Amaya, both in their protocol and in the Troubleshooting and Q&A sections of their web page (http://www.welc.cam.ac.uk/~ea3/Trouble.Shooting.Section.html and http://www.welc.cam.ac.uk/~ea3/Ques.Ans.Section.htm). We have found that additional modifications to the technique improves the efficiency of transgenesis. Here are some more useful improvements that we discovered the hard way.

Transgenic reaction mixture

• We use a total of 100-200 ng of plasmid to minimize the possibility of multiple insertions.
• Also, try to use equimolar amounts of DNA.  For example, in 100 ng of a 3000bp plasmid, there will twice as many plasmid molecules than in 100 ng of a 6000bp plasmid.  So use between 100-200 ng of the control plasmid (e.g. GFP) and use an equimolar amount of the plasmid carrying your gene of interest.
• We increased the dilution of SalI to 1:40
• We eliminated the magnesium because it appeared to be detrimental to normal development. Magnesium does increase rate of transgenesis, but we found it more important to have normally developing transgenic animals rather than large numbers of abnormal transgenics.
• Use 10µl of HEATED egg extract for the reaction mixture.  The egg extract is heated at 80°C for 10 min and then centrifuged at 20,000 g for 2 min ( in microcentrifuge).  This modification, which was first devised by John Gerhart and Michael Wu, appears to remove factors in the extract. that impede development.  The resulting supernatant still contains nucleoplasmin (a histone binding nuclear protein), which is the active ingredient for chromosome decondensation.
• Throughout the procedure, be very gentle with the sperm nuclei, especially after they have been  decondensed with egg extract.

Transplantation needles

• The taper can be anywhere between 7 mm and 15 mm.
• Try to keep the bore size between 60µm to 80µm, as anything smaller can damage the sperm and anything bigger will damage the egg.
• Dimethyldichlorosilane can be used to coat the needles instead of Sigmacote.
• Make sure the needle is beveled approximately 45° to ensure that you can easily pierce the egg.

• 2% agarose works just as well as 2.5% (agarose is expensive).
• Avoid bubbles in the agarose when filling dishes. Otherwise, eggs will fall into the bubbles and will be difficult to inject.
• Dishes should not be reused more than once.  When storing for reuse, they MUST be dry, or the eggs will not stick well to the bottom of the dish.  For example, we would clean the agarose dishes with water and leave to dry on lab bench overnight for use the next day.

• Remove as much excess blood and fat from the testes as possible before macerating.
• Pipet the macerated testes up and down to further break up the pieces. Use an Eppendorf pipette with a cut blue tip.
• Reusable nylon mesh can be used to filter the testes instead of cheesecloth (which can't be reused). Nylon also filters better because the holes are finer (80 µm), and it won't absorb the sperm.
• Eliminate protease inhibitors from the sperm preparation.  i.e. NO leupeptin or PMSF.
• Replace the use of lysolecithin with digitonin, keeping the concentration and incubation time the same;  i.e., incubate sperm pellet resuspended in 1 ml of 1 X NPB for 5 min with 50 µl of 10 mg/ml digitonin.  Digitonin is plasma membrane specific because it targets cholesterol. Since there is no cholesterol in the nuclear membrane, this allows for demembranation with minimal nuclei damage.
• Sperm can be frozen in aliquots for storage.  Sperm can be quick frozen or slow frozen.  Quick freezing is done by dropping aliquoted tubes of sperm into liquid nitrogen.  The sperm can then be transferred to -80°C freezer for storage.  Slow freezing is done by placing aliquoted tubes of sperm into -20°C freezer, then transfering them to -80°C once frozen.  Before aliquoting and freezing, IT IS EXTREMELY IMPORTANT THAT THE SPERM BE MIXED VERY WELL IN SSB, because the glycerol in the SSB is what protects that sperm.
• Use a fresh aliquot of sperm every injection day.
• We use DAPI instead of Hoescht stock to stain the nuclei for counting.

• Eggs from younger frogs are more likely to produce healthy embryos than eggs from older frogs that have been spawned often.
• Inject females with HCG and leave them overnight (approx. 12-14 hr).
• When dejellying eggs, we use 2% cysteine because it is not as harsh on the egg membrane as 2.5%. Also don't leave the eggs in cysteine for too long.  Eggs from different females will require different amount of times for dejellying.  Time for less than 5 min and keep an eye on the eggs thereafter.  The eggs are dejellied when they begin to touch each other.
• Let the eggs sit in the 6% Ficoll for 5 min in the agarose dish before injecting, and they won't slide around during injection.

• Make sure there is liquid flowing out of the needle before you start injecting.  This should be easy to see because the sperm mixture flowing out of the needle is of a different viscosity than the Ficoll medium.
• If you hear a snapping sound, check to see if the needle is broken!
• The preceding two tips will save you the agony of injecting 500 eggs with nothing.
• Make sure you don't stab the needle through the other side of the egg. Eggs can tolerate one wound but usually not two.
• If the needle is clogged with cytoplasm, try raising it out of the liquid quickly before continuing.

• Store embyros at 18°C as soon as possible and keep them there until hatching.  Best if done IMMEDIATELY after microinjection.  DO NOT however, store at temperatures under 14°C.  Having an incubator set at 18°C is the easiest way.  But if an incubator is not available, try to be creative.  For example, you can try placing the embryos on top of a lidded container with a small amount of ice in it.  (remember: temperatures below 14°C will impair development).
• It is best to select normally cleaving embryos when they have reached the four- or eight-cell stage.
• We transfer the normally cleaving embryos to 0.1X MMR + 6% Ficoll + gentamycin right away, then transfer them to 0.1X MMR + gentamycin (no Ficoll) the next morning.
• Remove dead embryos regularly so they won't affect the normal ones.