-210 Nottingham - Nottingham eTheses - The University of Nottingham

The procedure is to expel the PBI and its adjacent cytoplasm through a slit made in
the zona pellucida by increasing the pressure inside the holding pipette. Porcine
clones have been produced by this method (Lee et al., 2003; Lee et al., 2008).
The advantage is that the oocytes do not need to be exposed to UV light. The
disadvantage is mainly that it is very complicated and there is little control over the
volume of cytoplasm extruded during the enucleation process (Li et al., 2004).
1.3.3.8 Other enucleation methods
Other methods for enucleation include bisection of oocytes, centrifugation, or
visualising DNA using fluorochrome Sybr14 or Pol-Scope microscopy (Li et al.,
2004).
1.3.4 Reconstruction strategies
The reconstructed embryos are made from donor cells and enucleated oocytes. The
strategies include single transfer, serial nuclear transfer and zona-free nuclear
transfer.
1.3.4.1 Single transfer
This protocol involves injection of an intact donor cell into the perivitelline space of
an enucleated MII oocyte, fusion was introduced by a brief period of electrical pulses,
followed by activation of oocyte-donor cell couplets (Wilmut et al., 1997; Betthauser
et al., 2000). This method is simple and basically follows the protocol by Campbell et
al. (1996b).
Alternatively, single transfer can be carried out by a distinctive nonfusion method. It
is to directly inject the nuclei of donor cells instead of the entire donor cells into
enucleated oocytes using the piezo-micromanipulation equipment and then activated
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