Target Discovery and Validation Reviews and Protocols

Keratin Transgenics and Knockouts 241
2. Wash aggregation needle briefly in ethanol.
3. Make about six aggregation wells into each of the drops by pressing and turning
the needle into the ground of the tissue culture dish.
4. Place aggregation plate in an incubator at 37°C, 5%CO 2 for at least 2 h.
3.11.5. Removal of Zona Pellucida With Tyrode’s Solution
The following supplemental materials are needed for these experiments:
dissecting microscope, acid Tyrode’s solution (cat. no. T1788, Sigma), tissue
culture dishes (100 × 15 mm), M2 and KSOM-AA medium, and mouth
pipet.
1. Add embryos to the first M2 drop. Rinse 20 embryos in the first drop of Tyrode’s
solution and transfer them into the second drop.
2. Observe dissolving of the zona pellucida with the microscope and instantly transfer
the embryos to the M2 drop containing 0.3% BSA to prevent embryos from
sticking to the plate or to each other.
3. Wash embryos by transferring them through 3 drops of M2 and 3 drops of
KSOM-AA.
4. Transfer embryos into a KSOM-AA drop without depressions on the aggregation
plate.
5. Trypsinize ES cells for aggregation if ES cell/tetraploid embryo aggregation is
planned.
3.11.6. ES Cells or Diploid Embryo/Tetraploid Embryo
“Sandwich” Aggregation (Table 3)
The following supplemental materials are needed for these experiments: dissecting
microscope, prepared aggregation plate with depressions and embryos
with removed zona, trypsinized ES cells or eight-cell embryos, and mouthcontrolled
pipet.
1. Transfer tetraploid embryos in 1 microdrop of the aggregation plate without
depressions.
2. Also transfer diploid eight-cell knockout embryos or trypsinized ES cells into
another microdrop.
3. Place a tetraploid embryo in each of the aggregation wells.
4. Place an eight-cell embryo or a clump of approx 10 ES cells on the tetraploid
embryo in each depression.
5. Place a second tetraploid embryo on top of the eight-cell embryo or ES cell
clumps, respectively, thus forming a sandwich.
6. Repeat steps 2 to 4 for all aggregations and culture the plate overnight in an incubator
at 37°C and 5% CO 2 .
7. On the afternoon of the next day check aggregates for blastocyst formation.
Transfer blastocysts into the uteri of day 2.5 p.c. pseudopregnant foster
females.