serum, 1% Penicillin-Streptomycin. Rat serum is commercially
available (for example Janvier) or can be homemade [8]. It is
best collected from males older than 12 weeks. If the commercial serum is delivered frozen, it should be gently thawed on
ice, aliquoted in small amounts (0.5–2 mL) and stored at
À80
C. Rat serum aliquots are heat-inactivated at 56
C for
30 min prior to preparation of the culture medium.
2.4 Mouse Lines
Mice colonies must be maintained in a certified animal facility in
accordance with local guidelines. The local ethics committee must
approve all experiments. The mouse lines used in this protocol are
the mT/mG [11] and the Sox2-Cre ER
T2 [12] lines available at the
Jax Laboratory (see Note 2). Rosa
mT/mG is a cell membranetargeted, two color fluorescent Cre-reporter allele. Cell membrane
tdTomato (mT) expression is universal, and Cre recombinase
expressing cells (and their descendants) have membrane-localized
EGFP (mG) expression replacing the red fluorescence. The Sox2Cre ER
T2 knock-in mice have the Sox2 open reading frame replaced
with a Cre ER
T2 fusion gene. Sox2 is expressed in the inner cell mass
at E3.5 then in the epiblast. Cre ER
T2 is a fusion of the Cre
recombinase with a mutant form of the human estrogen receptor
that does not bind its natural ligand at physiological concentration
but binds 4-hydroxytamoxifen. Cre ER
T2 can only gain access to
the nuclear compartment after exposure to tamoxifen. A portion of
epiblast cells proportional to the amount of tamoxifen will be
labeled with GFP, and those located in the primitive streak will
undergo EMT, allowing recording cell behavior as they exit the
primitive streak to become mesenchymal.
3 Methods
3.1 Mice
2–3 female mT/mG homozygous mice (at least 6 weeks old) are
placed in a cage with a single Sox2-CreER male (referred to as a
breeding triangle). Females are checked for the presence of a vaginal plug the following morning. The day of plug detection is
counted as embryonic day 0.5 (E0.5), since mating is assumed to
have occurred at the midpoint of the dark period. Pregnant females
are injected intraperitoneally with preheated (37
C) 0.1 mg/g
OH-tamoxifen (roughly 2 mg in 200 μL sesame oil) at E6.25 and
E6.75 (see Note 3) and euthanized by cervical dislocation at E7.25
for collection of embryos. The dosage of tamoxifen can be adjusted
to the degree of mosaicism one is trying to achieve.
3.2 Collection
of Embryos (Fig. 2)
1. Before starting the dissection, equilibrate and pre-warm the
culture medium by placing it in a tube slightly opened in a
humidified incubator at 37
C and 5% CO 2 in atmosphere for
at least 1 h. The dissection medium should be at room
temperature.
Ex vivo Imaging of EMT in the Early Mouse Embryo
139
available (for example Janvier) or can be homemade [8]. It is
best collected from males older than 12 weeks. If the commercial serum is delivered frozen, it should be gently thawed on
ice, aliquoted in small amounts (0.5–2 mL) and stored at
À80
C. Rat serum aliquots are heat-inactivated at 56
C for
30 min prior to preparation of the culture medium.
2.4 Mouse Lines
Mice colonies must be maintained in a certified animal facility in
accordance with local guidelines. The local ethics committee must
approve all experiments. The mouse lines used in this protocol are
the mT/mG [11] and the Sox2-Cre ER
T2 [12] lines available at the
Jax Laboratory (see Note 2). Rosa
mT/mG is a cell membranetargeted, two color fluorescent Cre-reporter allele. Cell membrane
tdTomato (mT) expression is universal, and Cre recombinase
expressing cells (and their descendants) have membrane-localized
EGFP (mG) expression replacing the red fluorescence. The Sox2Cre ER
T2 knock-in mice have the Sox2 open reading frame replaced
with a Cre ER
T2 fusion gene. Sox2 is expressed in the inner cell mass
at E3.5 then in the epiblast. Cre ER
T2 is a fusion of the Cre
recombinase with a mutant form of the human estrogen receptor
that does not bind its natural ligand at physiological concentration
but binds 4-hydroxytamoxifen. Cre ER
T2 can only gain access to
the nuclear compartment after exposure to tamoxifen. A portion of
epiblast cells proportional to the amount of tamoxifen will be
labeled with GFP, and those located in the primitive streak will
undergo EMT, allowing recording cell behavior as they exit the
primitive streak to become mesenchymal.
3 Methods
3.1 Mice
2–3 female mT/mG homozygous mice (at least 6 weeks old) are
placed in a cage with a single Sox2-CreER male (referred to as a
breeding triangle). Females are checked for the presence of a vaginal plug the following morning. The day of plug detection is
counted as embryonic day 0.5 (E0.5), since mating is assumed to
have occurred at the midpoint of the dark period. Pregnant females
are injected intraperitoneally with preheated (37
C) 0.1 mg/g
OH-tamoxifen (roughly 2 mg in 200 μL sesame oil) at E6.25 and
E6.75 (see Note 3) and euthanized by cervical dislocation at E7.25
for collection of embryos. The dosage of tamoxifen can be adjusted
to the degree of mosaicism one is trying to achieve.
3.2 Collection
of Embryos (Fig. 2)
1. Before starting the dissection, equilibrate and pre-warm the
culture medium by placing it in a tube slightly opened in a
humidified incubator at 37
C and 5% CO 2 in atmosphere for
at least 1 h. The dissection medium should be at room
temperature.
Ex vivo Imaging of EMT in the Early Mouse Embryo
139