Methods

M-phase synchronization:

1 Aspirate conditioned medium from the mES cells, replace with nocodazole-supplemented mES-cell medium, and incubate cells for 3-5 h.

2 Harvest individual mES cells in M-phase using a cell-transfer capillary under mouth control: M-phase (i.e. mitotic) mES cells are spherical, and easily visualized by light microscopy (Figure 2).

3 Pool and transfer M-phase mES cells into a droplet of nocodazole-supplemented M2 or FHM under oil, on the micromanipulation chamber, in preparation for nuclear transfer.

G1-phase synchronization (32):a

1 Prepare microdrops (5-10 ml) of aphidicoline-supplemented mES-cell medium under oil, in a culture dish; and of aphidicoline-supplemented M2 or FHM under oil, on the microinjection chamber.

2 Manually harvest M-phase mES cells (see steps 1 and 2, above), and wash through several 1 ml drops of nocodazole-free, aphidicoline-supplemented mES-cell medium.

3 Pool and transfer the M-phase mES cells into a final microdrop of aphidocoline-supplemented mES-cell medium, and culture for 50-60 min for cell division to become complete.

4 Collect the cleaved daughter cells and transfer them into a microdrop of aphidico-line-supplemented M2 or FHM on the microinjection chamber, in preparation for nuclear transfer.

aAn alternative, simpler method of harvesting Grphase mES cells is simply to prepare a single-cell suspension using trypsin/EDTA solution (Protocol 1), transfer cells into mES-cell medium, and select the smallest cells (which may represent the Grphase fraction) using a cell-transfer capillary and phase-contrast microscopy.

Experiments to ascertain the best cell-cycle combinations and method for mES cell-NT are still ongoing, and the heterogeneous nature of mES cells in culture (23) signifies that the precise efficiency of producing mES cell-derived mice may be largely dependent upon the particular cell line used and the conditions of its maintenance. However, it has been reported that the rate of development into blastocysts was considerably higher when M-phase mES cells were used as karyoplasts, compared with Grphase mES cells: 56.8-70.0% vs 5.9-14.1% by Zhou et al. (31); and 30-67% vs 2-5% by Yabuuchi et al. (27). Following transfer of such blastocysts into recipient females, full-term development was achieved at similar rates for both cell-cycle stages in the karyoplast (1.5-3.1% by Zhou et al. (31)), or was obtained only using M-phase karyoplasts (3-4%) (27).

5.3 Enucleation of Mil-phase oocytes

Recipient oocytes for nuclear transfer are collected at Mil-phase from superovu-lated, F1 hybrid female mice, for example B6C3F1 (C57BL/6 x C3H) or BDF1 (C57BL/6 x DBA). When cell-fusion methods are to be employed for nuclear transfer, the zona pellucida should be cut partially in preparation for enucleation. When the direct-injection method is to be used, the zona-cutting process is unnecessary; instead, the enucleation pipette is placed in contact with the surface of the zona pellucida, which it penetrates when Piezo-pulses are applied.

Figure 2 M-phase mES cells.

Cells were treated with 3 mg/ml nocodazole for 5 h. The rounded cells in this colony are blocked at metaphase.

Figure 2 M-phase mES cells.

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