Transgenesis in Xenopus
Improvements to the transgenic
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
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
- Use cut pipet tips and pipet slowly.
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
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.
- Don't get bubbles in the solution when pipetting
- Keep condensed nuclei on ice, but keep decondensed nuclei at room temperature.
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
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.
Egg collection and dejellying
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
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
Use a fresh aliquot of sperm every
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
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
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
If the needle is clogged with cytoplasm,
try raising it out of the liquid quickly before continuing.
This page created by members of the Browder Lab: Rob
Chan, Ivana Kostic and Pierrette Lo.
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
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.
July 29, 1999