Use Of Nuclear Transfer Research Articles

Transgenic protein production: Achievements using microinjection technology and the promise of nuclear transfer

Over the past decade the utility of the livestock mammary gland as a bioreactor for the production of recombinant human proteins has been clearly demonstrated. The benefits of transgenic protein production include safety from human viral contamination (e.g., HIV or hepatitis), low-cost, highvolume production, correct posttranslational modifications, and applicability to a wide range of complex proteins and peptides. PPL has expressed a number of human plasma-derived proteins in sheep at commercially useful levels, including fibrinogen, factor IX, and factor VII, and a nutritional protein, human α-lactalbumin, has been produced at high levels (2.4 g/L) in transgenic cows. PPL's lead product, human α-l-antitrypsin (AAT), has been produced at levels up to 35 g/L in sheep milk and is currently in phase II clinical trials for treatment of cystic fibrosis. With the advent of somatic cell nuclear transfer technology (cloning), we have seen the birth of Polly (our cloned sheep transgenic for factor IX) and a cloned bull calf, Mr. Jefferson. The use of nuclear transfer for production of transgenic animals has numerous benefits, including increased efficiency, shortened timelines, and the ability to make precise genomic modifications such as gene knockouts, which should open new avenues for transgenic protein production, as well as biomedical and agricultural applications.

Prospects for the use of nuclear transfer in human transplantation.

The successful application of nuclear transfer techniques to a range of mammalian species has brought the possibility of human therapeutic cloning significantly closer. The objective of therapeutic cloning is to produce pluripotent stem cells that carry the nuclear genome of the patient and then induce them to differentiate into replacement cells, such as cardiomyocytes to replace damaged heart tissue or insulin-producing beta cells for patients with diabetes. Although cloning would eliminate the critical problem of immune incompatibility, there is also the task of reconstituting the cells into more complex tissues and organs in vitro. In the review, we discuss recent progress that has been made in this field as well as the inherent dangers and scientific challenges that remain before these techniques can be used to harness genetically matched cells and tissues for human transplantation.

Review: Somatic Cell Nuclear Transfer in Mammals: Progress and Applications

Somatic nuclear transfer has been performed with frogs since the early 1960s, yet it has proved impossible to generate an adult frog using an adult cell as nuclear donor. After some initial skepticism, the birth of sheep, cows, goats, and mice using this technique with fetal or adult cell donors is now established fact. The success with adult mammalian cell donors extends the historic work in frogs by attesting to the totipotency of nuclei in at least some adult, differentiated cell types. Because the technique offers a developmental read out of the totality of genetic and molecular lifetime changes accumulated by the nucleus of a single somatic cell, basic research applications are seen in the fields of ageing, cancer, X chromosome inactivation, and imprinting. The prospect of a method for gene targeting in livestock holds particular promise for commercial applications; whilst for humans, the use of nuclear transfer to provide diverse populations of customized stem cells for therapeutic purposes presents a tantalizing future goal.

Therapeutic cloning: Needs and prospects

Introductionherapeutic cloningdescribes the use of nuclear transfer to provide cells, tissues, and organs for a patient requiring replacement or supplementation of diseased or damaged tissue. The same term has also been used to describe specific applications of human reproductive cloning, where the objective is to correct heritable abnormalities transmitted through the female mitochondria. In this article, we will restrict discussion to the potential application of cell cultures derived from disaggregated human pre-implantation embryos produced by nuclear transfer.Although it has long been recognised that human embryonic stem cells could possibly be used as a source of differentiated cells for human therapy,1,2two recent developments have triggered current excitement in this area. First, the derivation of pluripotent stem cells from human embryos3and human primordial germ cells.4Second, the success of nuclear transfer from adult somatic cells.5–7The suggestion has arisen that both scientific advances could be combined. Somatic cells could be obtained by biopsy, their nuclei transferred into an oocyte, human blastocysts derived and used to produce ES cells which are then induced to differentiate into the cell type required.8,9Transplant of such tissue would provide the patient with an autologous graft.$PThe prospect of therapeutic cloning in humans attracts less hostile comment than does reproductive cloning. Nevertheless, this is still a controversial area (see article in this issue by Burley), since the experimental production and culture of human blastocysts raises difficult ethical issues. In our view, any justification for its use must take account of possible therapeutic alternatives. Here we review these alternatives as well as the background and immediate prospects for therapeutic cloning.

Assessment of cytoplasmic competence of prepubertal calf oocytes by use of nuclear transfer

The objectives of this study were to evaluate the effect of sperm dose and sire on the fertilization rate, cleavage rate and blastocyst yield following insemination in vitro, to examine the relationship between these parameters and field fertility in cattle, and to examine the relationship between blastocyst quality and sire used in IVF. Frozen semen from four bulls with 150-day nonreturn rates ranging from 57 to 78% was used. In Experiment 1, oocytes were inseminated with sperm from one of the four bulls at concentrations ranging from 0.016 to 0.5×106 sperm/ml. A proportion of presumptive zygotes were fixed at 17 h post-insemination (hpi), while the remainder was transferred to in vitro culture (IVC) in droplets of synthetic oviduct fluid (SOF). Cleavage at 48 hpi and the percentage of oocytes reaching the blastocyst stage by Day 8 were recorded. In Experiment 2, to assess blastocyst quality, after insemination with semen from one of the four bulls, presumptive zygotes were cultured in SOF until Day 7. Blastocysts for each bull were removed and vitrified/warmed and survival was recorded at 24, 48 and 72 h after warming. Regardless of bull used, a concentration of 0.125×106 sperm/ml or above resulted in higher blastocyst yields than any lower concentration used. Fertilization and cleavage rates were also higher at higher sperm concentrations. The best predictor of field fertility was fertilization rate at a concentration of 0.5×106 sperm/ml (r=0.94, P<0.0001). There was also a significant correlation between cleavage rate at a concentration of 0.5×106 sperm/ml and nonreturn rate (r=0.90, P<0.0001). In Experiment 2, blastocysts derived from one bull, HTA, were of superior quality as measured by survival 24 h after thawing, although these differences were less significant at the subsequent time points measured. In conclusion, these data show that differences between the field fertility of bulls can be determined at sperm concentrations routinely used in IVF. Lowering the sperm concentration does not increase the likelihood of optimizing the differences in fertility or cleavage rate between bulls of different field fertility. We have also demonstrated that the bull can have a significant effect on the quality of blastocysts produced using IVF techniques.

Enucleation by centrifugation of in vitro-matured bovine oocytes for use in nuclear transfer.

Nuclear transfer has the potential to produce large numbers of identical progeny. Current limitations of the technique are associated with the use of micromanipulation for the demanding enucleation and reconstitution procedure. With the overcoming of this limitation, increased numbers of nuclear transfer embryos could be produced. Centrifugation of bovine oocytes at 15,000 x g for 2 min resulted in the stratification of organelles within the cytoplasm which positioned the metaphase II spindle for enucleation. After removal of the zona pellucida with Pronase, the oocytes were centrifuged in a Percoll density gradient so that the oocytes were stretched apart to form cytoplasts and the metaphase II spindle was separated from the majority of oocytes. Enucleation by centrifugation efficiently produced a consistent population of enucleated cytoplasts from bovine in vitro-matured oocytes. The population of enucleated cytoplasts was enriched by exclusion of the cytoplasts that exhibited an extrusion cone containing metaphase II chromosomes 6 h after centrifugation. The enucleated oocyte cytoplasts were aggregated with blastomeres isolated from in vivo-collected morulae. The aggregated embryonic cells were electrofused to obtain nuclear transfer embryos that were placed into a sodium alginate false zona and were capable of cleavage and development in vitro. The development of nuclear transfer embryos produced through use of centrifugation and aggregation techniques was comparable with that of nuclear transfer embryos produced by micromanipulation techniques.

Production of calves by transfer of nuclei from cultured inner cell mass cells.

We report here the isolation and in vitro culture of bovine inner cell mass (ICM) cells and the use of ICM cells in nuclear transfer to produce totipotent blastocysts that resulted in calves born. Of 15 cell lines represented in this study, 13 were derived from immunosurgically isolated ICM of 3 in vitro produced day 9-10 bovine blastocysts, while 2 lines were derived from single blastocysts. Approximately 70% of attempted cell lines became established cell lines when started from 3 ICMs. The ability to establish cell lines was dependent on the number of ICMs starting the line. Sire differences were noted in the ability of ICMs to establish cell lines and to form blastocysts. The cell lines were cultured as a low cell density suspension in the medium CR1aa plus selenium, insulin, and transferrin (SIT) and 5% fetal calf serum (FCS) for 6-101 days before use in nuclear transfer, at which time some had multiplied to more than 2000 cells. If allowed to aggregate, cells of established cell lines formed embryoid bodies. A total of 659 nuclear transfer clones were made by fusing the ES cells into enucleated oocytes with polyethylene glycol; 460 of these fused, based on cleavage (70%). After culture of the clones for 7 days in vitro in CR1aa/SIT/5% FCS, 109 (24%) of those fused became blastocysts. Thirty-four blastocysts were transferred into uteri of 27 cows, and 13 cows (49%) became pregnant. Four of the 13 cows gave birth to 4 normal calves. DNA typing showed the calves to be derived from the respective sires of the cell lines. The calves were derived from cultures of less than 28 days.

Production of genetically identical swine: Uses for families with reduced phenotypic variation

Recent advances in reproductive technology hold promise for future production of nearly unlimited numbers of swine with identical genotypes through the use of nuclear transfer. Potential benefits of use of these animals in the swine industry and in research are examined in the present study. Variability of litter size, days to slaughter and backfat thickness are predicted for clones, full-siblings and unrelated animals. One hundred simulation runs of 100 unrelated animals and 25 families of 4 full-siblings and clones were completed for each trait. Expected phenotypic standard deviations were 2.5 pigs for litter size, 12 d for days to slaughter and 2 mm for backfat thickness. Between family variances were expected to include 50% additive genetic variance, 25% dominance variance and 100% common environmental variance for full-siblings and 100% of all 3 components for clones. Substantial advantages of clones over unrelated animals and smaller advantages of clones over full-siblings were found for variability of days to slaughter and backfat thickness, but little difference was found for litter size. Ratios of error mean squares from analyses of variance suggest that use of clones rather than unrelated animals could reduce the number of animals needed for experiments by 67 and 65% for days to slaughter and backfat thickness, respectively, but only by 12% for litter size. These results suggest that the first use of cloned swine would be to reduce the numbers of research animals needed for intensive studies.