Target Discovery and Validation Reviews and Protocols

240 Vijayaraj, Söhl, and Magin
alternating current (AC) field is applied to allow the correct orientation of
embryos (enable, 1 or 2 V on the display). AC fields that are too high can
cause lysis. The following supplemental materials are needed for these
experiments: cell fusion instrument, two dissecting microscopes, M2 and
KSOM-AA media, 0.3 M mannitol and tissue culture dish with KSOM-AA
microdrops covered with oil (cat. no. M8410, Sigma), and mouth- or fingercontrolled
pipet.
1. Place slide with electrodes onto a 100-mm Petri dish by “fixing” it with two drops
of water.
2. Disperse a drop of M2 medium and mannitol over the slide with the electrodes and
1 drop of M2 medium under it.
3. Place a large drop of mannitol between the electrodes.
4. Connect the electrodes with the pulse generator and make sure that all settings are
correct according to the manufacturer.
5. Place all recovered two-cell embryos into the M2 microdrop.
6. Wash a group of approx 30 embryos in the mannitol drop and transfer them
between the electrodes into the mannitol.
7. Switch on the ac current and watch the embryos orientating through a microscope,
until the contact area of the two blastomeres is parallel to the electrodes. Wait until
all embryos are in the proper orientation and use a mouth pipet to abut nonorientated
embryos.
8. Apply the pulse to the embryos and immediately switch of the AC field.
9. Wash the embryos twice in KMSO media and transfer them into a KMSO drop
overlaid by light mineral oil and put them into the incubator at 37°C, 5% CO 2 .
10. Exchange mannitol solution to prevent water evaporation and mannitol crystallization
45 min after applying the pulse.
11. Check for perfectly fused embryos and put them into a drop of KSOM-AA with
oil overlay.
12. Repeat the pulse with unfused two-cell embryos. Culture tetraploid embryos
overnight at 37°C, 5% CO 2 .
13. After culturing for 24 h, use four-cell tetraploid embryos for the aggregation
experiment, and discard all other embryos.
3.11.4. Preparation of Aggregation Plate
The following supplemental materials are needed for these experiments:
dissecting microscope, sterile tissue culture dishes (35 × 10-mm Easy Grip;
cat. no. 3001-3, Falcon); 1-mL syringe with 26-gage 1/2 needle of KSOM-AA
medium, light mineral oil (embryo tested), and aggregation (darning) needle
(DN-09).
1. Dispense small drops of KSOM-AA medium (~15 µL each) on a 100 × 20-mm
tissue culture dish. Flood dish with light mineral oil, until all drops are covered
completely.