Method of Nuclear Transfer

Enucleation of mature unfertilized oocytes
A group of oocytes (usually 15-20 in number) was transferred into a droplet (about 10 μl) of HEPES-CZB containing 5 ?g/ml cytochalasin B, which had been previously placed in the operation chamber on the microscope stage. After each oocyte was immobilized by an oocyte-holding pipette, its zona pellucida was “drilled” by applying several piezo-pulses at the tip of an enucleation pipette (about 10 ?m in inner diameter). The Metaphase II chromosome-spindle complex, recognized as a translucent spot in the ooplasm, was drawn into the pipette with a small amount of accompanying ooplasm, and then gently pulled away from the oocyte until the cytoplasmic bridge was severed. After all oocytes in one group were enucleated (which took about 10 min), they were transferred into cytochalasin B-free CZB and kept there for up to 2 h at 37.5?C. The efficiency of enucleation was 100% as assessed by fixing and staining the oocytes.

Injection of somatic cell nuclei into enucleated oocytes
A donor cell was drawn in and out of the injection pipette until its nucleus was almost freed from cytoplasm. For cells with ‘tough’ plasma membranes (such as tail fibroblasts), a few piezo pulses were applied to break these membranes. After the ‘denuded’ nucleus was drawn deep into the pipette, the same pipette was used for subsequent cell enucleation. In a few minutes several naked nuclei were lined up within a single pipette. These nuclei were injected one by one into enucleated oocytes as described for spermatid nucleus injection except that all manipulations were performed at room temperature (24-26?C). Using this approach, nuclei were injected into oocytes within less than 10 min after denudation. Injected oocytes were transferred and kept in CZB at 37.5?C for 1-3 h before the activation treatment.

Oocyte activation and inhibition of extra polar body extrusion
When the reconstructed oocytes were left in CZB for 1-3 h (37.5?C), the injected nucleus within each oocyte was transformed into chromosomes. The oocytes were then incubated for 6-7 h in Ca2+-free CZB containing both 10 mM Sr2+ and 5 ?g/ml cytochalasin B. Sr2+ was used to activate oocytes16 and cytochalasin B prevented extrusion of chromosomes and polar body formation, assuring that all chromosomes of the somatic cell nucleus remained in the cytoplasm of the activated oocyte. Within each activated oocyte, chromosomes transformed into two or three pseudo-pronuclei, in most cases. An oocyte with at least one pseudo-pronucleus was considered normally activated, and these were washed and cultured in Sr2+ -and cytochalasin-free CZB until they reached the 2- to 8-cell or morulae/blastocyst stage.

Embryo transfer
Two- to eight-cell embryos (24 h or 48 h after onset of activation) were transferred into the oviducts of pseudo-pregnant foster mothers (CD-1, albino) which had been mated with vasectomized CD-1 males 1 day previously. Morulae/blastocysts (78 h after activation) were transferred into uteri of foster mothers mated with vasectomized males 3 days previously. In the first series of experiments (Table 1), recipient females were euthanized between 8.5 and 12.5 dpc and examined for the presence or absence of embryos. All the ‘dpc’ numbers in this paper refer to recipient females rather than embryos’ age. In three other experiments (Tables 2, 3 and 4), females were euthanized at 19.5 dpc and their uteri were examined for the presence of fetuses and implantation sites. Live fetuses, delivered by caesarean section, were raised by lactating foster mothers (CD-1).