Cloned mice derived from embryonic stem cell karyoplasts and activated cytoplasts prepared by induced enucleation.

Our objective was to induce enucleation (IE) of activated mouse oocytes to yield cytoplasts capable of supporting development following nuclear transfer. Fluorescence microscopy for microtubules, microfilaments, and DNA was used to evaluate meiotic resumption after ethanol activation and the effect of subsequent transient treatments with 0.4 micro g/ml of demecolcine. Using oocytes from B6D2F1 (C57BL/6 x DBA/2) donors, the success of IE of chromatin into polar bodies (PBs) was dependent on the duration of demecolcine treatment and the time that such treatment was initiated after activation. Similarly, variations in demecolcine treatment altered the proportions of oocytes exhibiting a reversible compartmentalization of chromatin into PBs. Treatment for 15 min begun immediately after activation yielded an optimized IE rate of 21% (n = 80) when oocytes were evaluated after overnight recovery in culture. With this protocol, 30-50% of oocytes were routinely scored as compartmentalized when assessed 90 min postactivation. No oocytes could be scored as such following overnight recovery, with 66% of treated oocytes cleaving to the 2-cell stage (n = 80). Activated cytoplasts were prepared by mechanical removal of PBs from oocytes whose chromatin had undergone IE or compartmentalization. These cytoplasts were compared with mechanically enucleated, metaphase (M) II cytoplasts whose activation was delayed in nuclear transfer experiments using HM-1 embryonic stem cells. Using oocytes from either B6D2F1 or B6CBAF1 (C57BL/6 x CBA) donors, the in vitro development of cloned embryos using activated cytoplasts was consistently inferior to that observed using MII cytoplasts. Live offspring were derived from both oocyte strains using the latter, whereas a single living mouse was cloned from activated B6CBAF1 cytoplasts.

Embryo development and establishment of pregnancy after embryo transfer in pigs: coping with limitations in the availability of viable embryos.

Embryo transfer and pregnancy maintenance strategies in pigs were evaluated with reference to situations in which limited numbers of viable embryos or micromanipulated embryos are available, such as pig cloning. Development of embryos with compromised zona pellucida was compared with development of embryos with intact zona pellucida. Micromanipulation had no effect on blastocyst production rates after development in vivo or in vitro, but development in vivo improved the number of embryos reaching the blastocyst stage. Transfer of embryos with compromised zona pellucida resulted in live piglets. Several hormone treatments to maintain pregnancy were tested in a model in which three embryos were transferred into unmated recipient gilts, compared with transfer of three embryos into mated recipients. None of the hormonal treatments resulted in pregnancy rates of more than 25% at term and no more than 9% of transferred embryos survived, in comparison with 50% of the mated recipients successfully carrying 25% of transferred embryos. Lastly, the developmental potential of parthenogenetic embryos was assessed and 62% of transferred embryos resulted in pregnancies, none of which continued beyond day 55 of gestation. After co-transfer of three fertilized embryos with 55-60 parthenogenetic embryos into each of six recipients, two live piglets were delivered. The results from the present study indicate that transfer of zona pellucida compromised embryos can yield litters of normal piglets. In addition, it was demonstrated in a model system involving the transfer of three fertilized embryos into mature gilts that hormonal pregnancy maintenance strategies support a low proportion of embryos to term. Lastly, the present study shows for the first time a comparably effective but novel alternative for pregnancy maintenance in the pig involving the co-transfer of parthenote embryos.

Deletion of the alpha(1,3)galactosyl transferase (GGTA1) gene and the prion protein (PrP) gene in sheep.

Nuclear transfer offers a cell-based route for producing precise genetic modifications in a range of animal species. Using sheep, we report reproducible targeted gene deletion at two independent loci in fetal fibro-blasts. Vital regions were deleted from the alpha(1,3)galactosyl transferase (GGTA1) gene, which may account for the hyperacute rejection of xenografted organs, and from the prion protein (PrP) gene, which is directly associated with spongiform encephalopathies in humans and animals. Reconstructed embryos were prepared using cultures of targeted or nontargeted donor cells. Eight pregnancies were maintained to term and four PrP-/+ lambs were born. Although three of these perished soon after birth, one survived for 12 days. These data show that lambs carrying targeted gene deletions can be generated by nuclear transfer.

Nuclear transfer in practice.

The technique of nuclear transfer (NT) allows the production of embryos, fetuses, and offspring from a range of embryonic, fetal, and adult derived cell types in a range of species. Successful development is dependent upon numerous factors, including type of recipient cell, source of recipient cell, method of reconstruction, activation, embryo culture, donor cell type, and donor and recipient cell cycle stages. The present review will discuss the uses of NT, the techniques presently available, and the factors affecting subsequent development.

The effects of cell size and ploidy on cell allocation in mouse chimaeric blastocysts.

In a previous study of mouse tetraploid<-->diploid chimaeric blastocysts, tetraploid cells were found to be more abundant in the trophectoderm than the inner cell mass (ICM) and more abundant in the mural trophectoderm than the polar trophectoderm. This non-random allocation of tetraploid cells to different regions of the chimaeric blastocyst may contribute to the restricted tissue distribution seen in post-implantation stage tetraploid<-->diploid chimaeras. However, the tetraploid and diploid embryos that were aggregated together differed in several respects: the tetraploid embryos had fewer cells and these cells were bigger and differed in ploidy. Each of these factors might underlie a non-random allocation of tetraploid cells to the chimaeric blastocyst. A combination of micromanipulation and electrofusion was used to produce two series of chimaeras that distinguished between the effects of cell size and ploidy on the allocation of cells to different tissues in chimaeric blastocysts. When aggregated cells differed in cell size but not ploidy, the derivatives of the larger cell contributed significantly more to the mural trophectoderm and polar trophectoderm than the ICM. When aggregated cells differed in ploidy but not cell size, the tetraploid cells contributed significantly more to the mural trophectoderm than the ICM. In both experiments the contributions to the polar trophectoderm tended to be intermediate between those of the mural trophectoderm and ICM. These experiments show that both the larger size and increased ploidy of tetraploid cells could have contributed to the non-random cell distribution that was observed in a previous study of tetraploid<-->diploid chimaeric blastocysts.

Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts.

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.

Sheep cloned by nuclear transfer from a cultured cell line.

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.

Nuclear-cytoplasmic interactions during the first cell cycle of nuclear transfer reconstructed bovine embryos: implications for deoxyribonucleic acid replication and development.

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.

A comparison of rate and uniformity of embryo development in Meishan and European white pigs.

A comparison was made of the rate and uniformity of development of embryos recovered from Meishan and European white sows. The time of ovulation was estimated to be 34.3 and 49.0 h after the onset of oestrus in large white and Meishan sows, respectively. Embryos were recovered from a total of 38 Meishan and 37 European pigs between 18 and 219 h after the estimated time of ovulation. Embryos recovered after 18-59 or 44-82 h were classified into one of 11 stages (from early fertilization to early blastocyst), and the maximum blastocyst diameter was measured for embryos recovered 140-219 h after ovulation. There was no evidence of a difference between the genotypes in the stage or size of embryos at these times or of large differences between the genotypes in the extent of variation in embryo stage within females, although a minority of European white females had very variable embryos. As the differences between the embryos of the Meishan and the European white were small, it seems unlikely that greater uniformity of Meishan embryo development is a major cause of the higher prenatal survival in that breed.

A clinical assessment of real time ultrasound.

Clinical experilence with a real time ultrasound machine (ADR 2130, Kretztechnik (U.K.) Ltd.) has demonstrated its versatility and accuracy in early and late pregnancy. Fetal movements and circulation and the site of the placenta can be reliably and convincingly displayed. The assessment of the biparietal diameter is at least as accurate as similar measurements made with a compound sector scan instrument. An important advantage in clinical practice of the ADR 2130 is its portability.