Reagents and equipment

Enucleated oocytes (Protocol 8)

Single-cell suspension of mES cells in either M2 or FHM (Protocol 1)

PVP-medium: M2 or FHM in which BSA is replaced with 10% polyvinyl pyrrolidone (PVP; Cat. No. PVP-360, Sigma)a

1 Microinject single karyoplasts into the perivitelline space of cytoplasts.

2 Equilibrate the karyoplast-cytoplast complexes in electrofusion medium, i.e. until they sink.

3 Place a flat drop of electrofusion medium over the central portion of the platinum electrodes in the electrofusion chamber, and transfer into it the reconstituted oocytes.

4 Orient the complexes relative to the platinum electrodes so that the karyoplasts are parallel to the wires, using an embryo-transfer capillary under mouth control.

5 Deliver two DC pulses of 50 V/mm for 50 ms with a 2 s interval. Repeat the pulses after 20min.

6 Wash complexes by passing through drops of M16 or KSOM, and incubate in microdrops of the same. Monitor fusion occurring between karyoplasts and cyto-plasts over 1 h.

7 Prior to activation, incubate the fused complexes in cytochalasin B-supplemented M16 or KSOM for 1 h, to prevent the dispersal of the chromosomes.

a For the electrofusion of mES cells with oocytes, calcium must be absent from the medium to avoid the premature activation of oocytes.

• PVA-medium: M2 or FHM in which BSA is replaced with 10% polyvinyl alcohol (PVA; P8136, Sigma)a ta unit e.g. the Piezo Impact Micromanipulator (Cat. No. PMM-150FU, Prime Tech Ltd)

• Embryo- and cell-transfer capillaries • Light mineral oil (Protocol 2) (Protocol 3)

• Micromanipulator assembly (Protocol 5) adapted with a Piezo-driven microinjection

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