-210 Nottingham - Nottingham eTheses - The University of Nottingham

2.5.5 Electrofusion
Immediately after cell transfer, electrofusion was conducted on a heated stage at 39°C.
Each oocyte/cell couplet was rinsed once in 1 ml fusion medium (0.28 M mannitol,
0.05 mM MgC12, and 0.001 mM CaC12), and then placed between the electrodes in
200 gl fusion medium and aligned so that the contact surface between the cytoplast
and the donor cell was parallel to the electrodes. 2 consecutive 50-gsec DC pulses of
1.4 kV/cm were given using an Eppendorf Multiporator (Eppendorf, Hamburg,
Germany). To maintain constant temperature and concentration, the fusion medium
was regularly replaced. The pulsed couplets were removed from the fusion medium
and placed in PZM/1O% FBS medium at 39°C in a humidified atmosphere of 5%
CO2 in air until 44 hpm.
2.6 Caffeine treatment
Selected TI and early MII oocytes or TI enucleated oocytes were washed three times
in modified NCSU-23 (or HEPES-NCSU-23) and cultured in modified NCSU-23 (or
HEPES-NCSU-23) at 39°C in a humidified atmosphere of 5% CO2 in air until 38
hpm. Then they were washed three times in modified NCSU-23 with different
concentrations of caffeine (0,5,10 and 20 mM) and cultured in modified NCSU-23
with different concentrations of caffeine (0,5,10
humidified atmosphere of 5% CO2 in air until 44 hpm.
and 20 mM) at 39°C in a
2.7 Activation
For parthenogenetic activation, in vitro maturated oocytes were denuded of cumulus
cells by repeated pipetting on a warm stage at 39°C at 44 hpm. Or, oocytes were
denuded and treated with caffeine until 44 hpm as described in sections 2.4 and 2.6.
Denuded oocytes were selected, rinsed once in 1 ml activation medium (0.28 M
mannitol, 0.05 mM MgC12, and 0.1 mM CaC12) and placed between the electrodes in
the fusion chamber in 200 µl activation medium. They were activated by
administration of 2 consecutive 60-µsec DC pulses of 1.2 kV/cm. Activated oocytes
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