125extract and whole oocytes, and how to analyze targets of execu-
tioner caspase activation including DNA fragmentation and
poly(ADP-ribose) polymerase (PARP) cleavage.Mature female X. laevis should be housed in appropriate condi-
tions with tightly controlled water parameters. We house our frogs
in a multi-rack system from Aquatic Habitats ( http://www.
aquatichabitats.com ). The water must be maintained at 18 °C, at a
pH 7.2, and a conductivity 2 mS. This system is expensive; how-
ever, less sophisticated and cheaper methods of housing X. laevis
have also been used with success ( see Note 1 ). There are two injec-
tions that must be performed to induce egg laying by X. laevis
frogs. The fi rst is an injection of 0.6 mL PMSG (200 U/mL stock),
3–7 days prior to the second injection, and the desired time for
eggs to be laid. This “primes” the frog, inducing production and
maturation of more eggs ( see Note 2 ). The second injection is an
injection of 0.6 mL of hCG (1,000 U/mL stock), the day before
you wish the eggs to be laid ( see Note 3 ). This injection will induce
the frog to lay eggs, which can be collected the next morning. We
inject intramuscularly in the upper quadriceps of the frog’s leg.
Mature X. laevis eggs are arrested in metaphase of meiosis II,
due to high cyclin B/cdc2 activity. Lysis of the eggs by centrifuga-
tion induces a release of calcium from intracellular stores, which in
addition to the CaCl 2 in the egg lysis buffer induces activation of
calcium/calmodulin regulated protein kinase (CaMKII) which
leads to the degradation of cyclin B and entry into interphase of
mitosis. The egg extract is then maintained in interphase by the
addition of cycloheximide to the egg lysis buffer, preventing the
synthesis of new proteins, namely, cyclin B ( see Note 4 ).
Wash and de-jelly eggs- Collect the freshly laid eggs in a 200-mL glass beaker, and
pour off any excess water. - Prepare solutions of 2 % L-cysteine pH 8, 1× MMR, and 1×
ELB. For 1× ELB, include DTT at a fi nal concentration of
1 mM and sucrose to a fi nal concentration of 250 mM. - Wash and incubate the eggs in 2 % L-cysteine, pH 8 for approx-
imately 5 min, swirling the eggs periodically. Use approximately
50 mL of 2 % cysteine per frog. Xenopus eggs are surrounded by
a transparent jelly coat, which prevents the eggs from contact-
ing each other, that is removed by cysteine. When the jelly coat
is removed, the eggs will pack closely together ( see Note 5 ). - When the eggs are de-jellied, pour off the cysteine, avoiding
pouring off any eggs at this stage ( see Note 6 ). - Wash eggs two times in 1× MMR. For washes, briefl y swirl the
eggs and then pour off as much MMR as possible without
pouring off any of the eggs.
3.1 Collection
of X. laevis Eggs
and Preparation of
Interphase Egg Extract
Methods for the Study of Caspase Activation in the Xenopus laevis Oocyte...