Method

1 To facilitate micromanipulation, we recommend placing a column of three separate microdrops (~10 ml) on the micromanipulation chamber, the top microdrop contains karyoplasts in M2 or FHM either supplemented with nocodazole for the M-phase donor cells, or without nocodazole for G1/G0-phase donor cells; the middle microdrop, a suspension of inactivated Sendai virus; and the bottom microdrop, enucleated recipient oocytes (cytoplasts) in M2 or FHM. Cover the droplets with mineral oil.

2 From the top microdrop take up a single karyoplast into the microinjection pipette, transfer the pipette into the second drop, and draw up (by ~20 mm into the pipette) a small volume of the viral suspension.

3 Transfer the injection pipette into the third microdrop and inject its contents (kar-yoplast and virus suspension) into the perivitelline space of an enucleated oocyte, through the hole in the zona pellucida made originally for enucleation (Figure 4A, B).

4 Using the injection pipette gently push the karyoplast into direct contact with the oolemma of the cytoplast, to promote cell fusion (Figure 4C).

Na2HPO4.12H2O, and 13 mM Tris base in double-distilled water Ca2 + -supplement for BSS: a stock solution of 200 mM CaC12.2H2O in double-distilled water is diluted to a working solution of 2 mM in BSS

Light mineral oil or silicon oil (Protocol 2)

M16 or KSOM (Protocol 2)

Cytochalasin B-supplemented medium: 5 mg/ml cytochalasin B (Protocol 8) in M16 or KSOM

Embryo-transfer capillary (Protocol 3) Micromanipulator assembly (Protocol 5)

Protocol 9 continued

5 Repeat the micromanipulations with the remaining cytoplasts.

6 Using a dissecting microscope and an embryo-transfer capillary, collect and transfer all the manipulated embryos (i.e. karyoplast-cytoplast complexes) into fresh M16 or KSOM, wash several times in 1ml drops of the same medium, and incubate in microdrops.

7 Periodically monitor the complexes for fusion of karyoplasts with cytoplasts over 1 h of incubation, and select the fused complexes for activation (Protocol 12)a a Optimally, fusion should occur within 1 h of viral infection. Reduce the titre of the virus if cell lysis occurs, or increase it if the fusion rate is low. Aim for a fusion rate of more than 60%.

(B)
(C)
Figure 4 Sendai virus-induced fusion of karyoplast and cytoplast. Stages (A) to (C) of the procedure are explained in Protocol 9. Arrow indicates position of karyoplast.
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