Fertilization of mammalian eggs is followed by successive cell divisions and progressive differentiation, first into the early embryo and subsequently into all of the cell types that make up the adult animal. Transfer of a single nucleus at a specific stage of development, to an enucleated unfertilized egg, provided an opportunity to investigate whether cellular differentiation to that stage involved irreversible genetic modification. The first offspring to develop from a differentiated cell were born after nuclear transfer from an embryo-derived cell line that had been induced to become quiescent. Using the same procedure, we now report the birth of live lambs from three new cell populations established from adult mammary gland, fetus and embryo. The fact that a lamb was derived from an adult cell confirms that differentiation of that cell did not involve the irreversible modification of genetic material required for development to term. The birth of lambs from differentiated fetal and adult cells also reinforces previous speculation that by inducing donor cells to become quiescent it will be possible to obtain normal development from a wide variety of differentiated cells.

Nuclear transfer has been used in mammals as both a valuable tool in embryological studies and as a method for the multiplication of 'elite' embryos. Offspring have only been reported when early embryos, or embryo-derived cells during primary culture, were used as nuclear donors. Here we provide the first report, to our knowledge, of live mammalian offspring following nuclear transfer from an established cell line. Lambs were born after cells derived from sheep embryos, which had been cultured for 6 to 13 passages, were induced to quiesce by serum starvation before transfer of their nuclei into enucleated oocytes. Induction of quiescence in the donor cells may modify the donor chromatin structure to help nuclear reprogramming and allow development. This approach will provide the same powerful opportunities for analysis and modification of gene function in livestock species that are available in the mouse through the use of embryonic stem cells.

Since the first report of live mammals produced by nuclear transfer from a cultured differentiated cell population in 1995 (ref. 1), successful development has been obtained in sheep, cattle, mice and goats using a variety of somatic cell types as nuclear donors. The methodology used for embryo reconstruction in each of these species is essentially similar: diploid donor nuclei have been transplanted into enucleated MII oocytes that are activated on, or after transfer. In sheep and goat pre-activated oocytes have also proved successful as cytoplast recipients. The reconstructed embryos are then cultured and selected embryos transferred to surrogate recipients for development to term. In pigs, nuclear transfer has been significantly less successful; a single piglet was reported after transfer of a blastomere nucleus from a four-cell embryo to an enucleated oocyte; however, no live offspring were obtained in studies using somatic cells such as diploid or mitotic fetal fibroblasts as nuclear donors. The development of embryos reconstructed by nuclear transfer is dependent upon a range of factors. Here we investigate some of these factors and report the successful production of cloned piglets from a cultured adult somatic cell population using a new nuclear transfer procedure.

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.

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into any of the possible outcomes because of the Wada property.We have verified the Wada property experimentally for our laboratory model by using a beam of 0.48 mm diameter from a 0.95 mW helium-neon laser. If the beam is shone on a boundary point, we find that the laser light can be seen through each of the openings. However, if the beam is aimed at the interior of one of the coloured regions, the light is seen through only one opening, casting a bright spot on the corresponding poster board.We believe that Wada boundaries should be a typical feature in chaotic scattering systems that have more than two exit modes. We have verified the existence of Wada boundaries in a situation involving chemical reactions 4 with two and three degrees of freedom.

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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.