William A. Ritchie

Ovine primary fetal fibroblasts were cotransfected with a neomycin resistance marker gene (neo) and a human coagulation factor IX genomic construct designed for expression of the encoded protein in sheep milk. Two cloned transfectants and a population of neomycin (G418)-resistant cells were used as donors for nuclear transfer to enucleated oocytes. Six transgenic lambs were liveborn: Three produced from cloned cells contained factor IX and neo transgenes, whereas three produced from the uncloned population contained the marker gene only. Somatic cells can therefore be subjected to genetic manipulation in vitro and produce viable animals by nuclear transfer. Production of transgenic sheep by nuclear transfer requires fewer than half the animals needed for pronuclear microinjection.

The present study investigated the decay of maturation-promoting factor (MPF) activity in electrically activated in vitro-matured bovine oocytes and examined the influence of the cell cycle stage of both the donor nucleus and the recipient cytoplasm upon the morphology and DNA synthesis potential of the donor nucleus in reconstructed embryos. The decay of MPF activity was studied both biochemically in electrically activated in vitro-matured oocytes and by morphological examination of nuclear structure in reconstructed bovine embryos. As measured by H1 kinase activity in groups of 10 oocytes, the level of MPF declined rapidly to 30 +/- 4% (of the maximum level in unactivated control oocytes) at 60 min and reached a basal level of 20 +/- 6% at 120 min. This level of activity was then maintained until at least 9 h postactivation. In contrast, when MPF activity was assayed by morphological examination of nuclei in individual reconstructed embryos, the decline in activity occurred over a period of 9 h postactivation. DNA synthesis of nuclei arrested at the G1/S border and in G2 phases of the cell cycle was examined in embryos reconstructed at the time of oocyte activation, i.e., when MPF levels were maximal, and by fusion 10 h postactivation, when no MPF activity could be detected. All nuclei transferred at the time of oocyte activation underwent nuclear envelope breakdown (NEBD) and subsequent DNA synthesis. However, when nuclei were transferred 10 h after activation, no NEBD was observed in any nuclei. Nuclei arrested at the G1/S border or nuclei in S phase when transferred in the absence of NEBD underwent DNA synthesis, while no DNA synthesis was observed in G2 nuclei transferred into this cytoplasmic environment. The results presented show that all nuclei, regardless of cell cycle stage, undergo DNA replication when transplanted into metaphase II (MeII) cytoplasts in which MPF activity is high. From these observations we would suggest that one factor that may contribute to the present low frequency of development of bovine nuclear transfer embryos is the ploidy of the reconstructed embryo after the first cell cycle. In order to maintain correct ploidy of the reconstructed embryo, only G1 nuclei should be transferred at the time of activation, when MPF levels are high. In contrast, when the integrity of the nuclear membrane is maintained by transfer at 10 h postactivation, when MPF activity is absent, the rereplication of G2 nuclei is prevented and correct ploidy of the reconstructed embryo may be maintained.