-210 Nottingham - Nottingham eTheses - The University of Nottingham

transferred into 500 µl modified NCSU-23 containing 0.05 M sucrose (Fisher
Scientific) for selection at 39°C. Oocytes with an extrusion cone or polar body were
regarded as TI or early MIT oocytes. Selection of TI oocytes was also carried out in
enucleation medium in the microinjection chamber directly after cumulus removal.
2.5 Nuclear transfer (Campbell et al., 2006; Polejaeva et al.,
2005)
2.5.1 Instruments for micromanipulation
2.5.1.1 Preparation of holding pipettes
A 1.0 mm (o. d. ) x 0.58 mm (i. d. ) x 10 cm glass capillary (Intracel, England) was
pulled by hand over a small flame to make a long parallel length of glass of
approximately 150 mm. The pulled capillary was cut using a diamond pencil to give a
pipette with a 100-150 µm outer diameter (Figure 2.1). The end of the pipette was fire
polished over the filament of the microforge to close the end to a diameter of
approximately 25 µm. Finally, the pipette was positioned horizontally and bent about
1 cm from the tip at an angle of 30°, thus allowing the tip to be parallel to the
manipulation chamber when mounted in the micromanipulator (Burleigh Instruments
Inc., UK) attached to the microscope (Leica DMIRBE, Heidelberg, Germany).
2.5.1.2 Preparation of enucleation/nuclear transfer pipettes
A 1.0 mm (o. d. ) x 0.80 mm (i. d. ) x 10 cm glass capillary (Intracel, England) was
pulled using a moving-coil microelectrode puller (P-97, Sutter Instruments Co., USA)
to give a inner diameter of slightly more than the required diameter (e. g. 20 gm). The
capillary was mounted in the microforge and broken at the required diameter by
fusing the glass onto the glass bead on the microforge (MF-830, Narishige, Japan)
and turning off the heat while drawing it away. The pipette was then ground using a
microgrinder (EG-400, Narishige, Japan) at an angle of 45°. The tip was mounted in
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