1 Dispense into the micromanipulation chamber an upper row of two microdrops (5-10 ml) of PVP medium, and a lower row of two microdrops of PVA medium; and cover with mineral oil. The upper two microdrops of PVP medium are for manipulating karyoplasts (the first is reserved for clearing the microinjection pipette if it becomes obstructed with viscous material during micromanipulation, and the second is for containing the karyoplasts). The lower two microdrops of PVA medium are for direct injection (the third is for washing the karyoplasts, and the fourth for the process of direct injection).

2 Place the enucleated oocytes (cytoplasts) into the fourth microdrop of PVA-medium, in preparation for direct injection.

3 Using a cell-transfer capillary, place 100-200 mES cells into the second microdrop of PVP-medium. Repeatedly draw the cells up and down the microinjection pipette in order to destroy their plasma membranes.b

4 Rinse the microinjection pipette in the third microdrop (to avoid PVP contamination), and place the karyoplasts into the fourth microdrop of PVA-medium together with the enucleated oocytes, ready for direct injection. Secure an oocyte to the holding pipette and insert the microinjection pipette into the perivitelline space, through the same hole as was made for enucleation.

5 Push the microinjection pipette into the ooplasm as deeply as possible (Figure 5A). At this point the oocyte becomes distorted in shape.

6 Deliver a Piezo-pulse, according to the equipment manufacturer's instructions, to enter the oolemma (Figure 5B). Once the microinjection pipette breaks through the oolemma and into the ooplasm, and the round shape of the oocyte is restored, inject the karyoplast into the oocyte (Figure 5C).

7 Pool and transfer successfully injected oocytes into microdrops of M16 or KSOM; and, prior to activation, culture in cytochalasin B-supplemented M16 or KSOM for 1 h to allow recovery of the oolemma.

a Alternatively a complete formulation for Hepes-buffered KSOM, from which BSA may be omitted and PVP or PVA added, is given is Protocol 8, Chapter 2.

bThe diameter of the microinjection pipette should be 5-10 mm, i.e. just smaller than the diameter of the mES cell.

Figure 5 Direct injection of karyoplast into cytoplast by Piezo-driven micromanipulation. Details of the procedure are given in Protocol 11. (A) A donor cell is injected into the cytoplast, in the deepest area. (B) Piezo pulses are applied. (C) The karyoplast has been successfully injected.

with a fluorescent, DNA-binding dye such as Hoechst 33342, and observing the oocyte by fluorescence microscopy. It is also practical to perform enucleation simultaneously with fluorescence detection, provided that the exposure time of the oocyte to fluorescence illumination and observation is no longer than 10 s. Longer exposures result in damage specifically to the mitochondrial genome, rendering the oocytes unsuitable to act as cytoplasts.

5.4 Activation of nuclear-transfer embryos

Reconstituted, nuclear-transfer embryos require artificial activation to induce their development. Of the several methods available, chemical activation (with strontium chloride) and electrical activation are most commonly used for mouse nuclear-transfer embryos. Following successful fusion and activation, as denoted by pronucleus formation, nuclear-transfer embryos reaching the morula or blastocyst stages are transferred to the oviducts of day 1 p.c. pseudo-pregnant recipients. Live-born offspring may be delivered by Caesarean section and cross fostering on day 19p.c., or by natural birth at term. Alternatively, recipients may be sacrificed earlier to examine fetal development.

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