Science and technology of farm animal cloning: state of the art

Details of the first mammal born after nuclear transfer cloning were published by Steen Malte Willadsen in 1986. In spite of its enormous scientific significance, this discovery failed to trigger much public concern, possibly because the donor cells were derived from pre-implantation stage embryos. The major breakthrough in terms of public recognition has happened when Ian Wilmut et al. [Wilmut, I., Schnieke, A.E., McWhir, J., Kind, A.J., Campbell, K.H., 1997. Viable offspring derived from fetal és adult mammalian cells. Nature 385, 810-813] described the successful application of almost exactly the same method, but using the nuclei of somatic cells from an adult mammal, to create Dolly the sheep. It has become theoretically possible to produce an unlimited number of genetic replicates from an adult animal or a post-implantation foetus. Since 1997 a number of different species including pigs, goats, horses, cats, etc. have been cloned with the somatic cell nuclear transfer technique. Although the technology still has relatively low success rates and there seems to be substantial problems with the welfare of some of the cloned animals, cloning is used both within basic research and the biomedical sector. The next step seems to be to implement cloning in the agricultural production system and several animals have been developed in this direction. This article reviews the current state of the art of farm animal cloning from a scientific and technological perspective, describes the animal welfare problems and critically assess different applications of farm animal cloning. The scope is confined to animal biotechnologies in which the use of cell nuclear transfer is an essential part and extends to both biomedical and agricultural applications of farm animal cloning. These applications include the production of genetically identical animals for research purposes, and also the creation of genetically modified animals. In the agricultural sector, cloning can be used as a tool within farm animal breeding. We do not intend to give an exhaustive review of the all the literature available; instead we pinpoint issues and events pivotal to the development of current farm animal cloning practices and their possible applications.     

Cloning of Homo sapiens? No!

Animal cloning by nuclear transplantation was first developed in the northern leopard frog, Rana pipiens. It was soon extended to other amphibian species and within time, to various mammalian species. The production of a cloned sheep (Dolly) from an adult nuclear donor reawakened interest in human cloning. Nuclear transfer for the production of animal clones has served experimental biology well. Nonetheless, the potential burden of developmental hazards, scientists and funds diverted from more needy causes, as well as the potential assault on the concept of family has led the author to oppose human cloning.     

Somatic cell nuclear transfer: Past, present and future perspectives

It is now over a decade since the birth, in 1996, of Dolly the first animal to be produced by nuclear transfer using an adult derived somatic cell as nuclear donor. Since this time similar techniques have been successfully applied to a range of species producing live offspring and allowing the development of transgenic technologies for agricultural, biotechnological and medical uses. However, though applicable to a range of species, overall, the efficiencies of development of healthy offspring remain low. The low frequency of successful development has been attributed to incomplete or inappropriate reprogramming of the transferred nuclear genome. Many studies have demonstrated that such reprogramming occurs by epigenetic mechanisms not involving alterations in DNA sequence, however, at present the molecular mechanisms underlying reprogramming are poorly defined. Since the birth of Dolly many studies have attempted to improve the frequency of development, this review will discuss the process of animal production by nuclear transfer and in particular changes in the methodology which have increased development and survival, simplified or increased robustness of the technique. Although much of the discussion is applicable across species, for simplicity we will concentrate primarily on published data for cattle, sheep, pigs and mice.     

Nuclear equivalence, nuclear transfer, and the cell cycle

The last 20 years have seen the development of techniques for the production of mammals by nuclear transfer. Originally limited to the swapping of pronuclei and the use of early cleavage-stage embryos as nuclear donors, nuclear transfer came of age in 1995 with the birth of 2 Welsh Mountain lambs, Megan and Morag, that were produced using cultured differentiated cells as donors of genetic material. In 1996, Dolly was the first animal to be produced using the genetic material from an adult-derived somatic cell. The techniques used in the production of these animals have now been reproduced in both sheep and cattle, and as predicted, successful development has been obtained using donor cells taken directly ex vivo. This article reviews the current status of mammalian nuclear transfer and the biological background to these successes.     

Somatic cell nuclear transfer: infinite reproduction of a unique diploid genome

In mammals, a diploid genome of an individual following fertilization of an egg and a spermatozoon is unique and irreproducible. This implies that the generated unique diploid genome is doomed with the individual ending. Even as cultured cells from the individual, they cannot normally proliferate in perpetuity because of the "Hayflick limit". However, Dolly, the sheep cloned from an adult mammary gland cell, changes this scenario. Somatic cell nuclear transfer (SCNT) enables us to produce offspring without germ cells, that is, to "passage" a unique diploid genome. Animal cloning has also proven to be a powerful research tool for reprogramming in many mammals, notably mouse and cow. The mechanism underlying reprogramming, however, remains largely unknown and, animal cloning has been inefficient as a result. More momentously, in addition to abortion and fetal mortality, some cloned animals display possible premature aging phenotypes including early death and short telomere lengths. Under these inauspicious conditions, is it really possible for SCNT to preserve a diploid genome? Delightfully, in mouse and recently in primate, using SCNT we can produce nuclear transfer ES cells (ntES) more efficiently, which can preserve the eternal lifespan for the "passage" of a unique diploid genome. Further, new somatic cloning technique using histone-deacetylase inhibitors has been developed which can significantly increase the previous cloning rates two to six times. Here, we introduce SCNT and its value as a preservation tool for a diploid genome while reviewing aging of cloned animals on cellular and individual levels.     

硬骨鱼类细胞核移植的研究进展

自１９９７年世界首例体细胞核移植绵羊“多莉”诞生以来,动物细胞核移植引起世界范围内人们的关注。硬骨鱼类具有很多优点,是研究核移植良好的材料。作者综述了硬骨鱼类细胞核移植技术的发展和完善以及在核质互作、细胞核发育全能性、鱼类育种及纯系建立等方面的应用,并对鱼类核移植存在的问题和前景进行了概括。     

Amphibian and mammal somatic-cell cloning: different species, common results?

Since the production of Dolly the sheep cloning methods for somatic cells have been thoroughly described and are becoming routine. However, the rate at which live clones are produced remains low in all mammalian species tested so far. Remarkably, irrespective of the cloning protocol or the donor-cell type, all clones display common abnormalities, particularly in the placenta. The process is also complicated by early mortality of somatic-cell clones and the founder mammalian clone, Dolly the sheep, died in February 2003 aged six years. Based on published data and on our own experience, our view is that mammalian somatic-cell cloning and the pioneer nuclear-transfer data from amphibians have much in common. We suggest that the only way to improve nuclear reprogramming is to modify the chromatin structure of somatic cells before nuclear transfer, to provide the oocyte with a chromosomal structure that is more compatible with the natural reprogramming machinery of the oocyte.     

Human cloning: category, dignity, and the role of bioethics

Human cloning has been simultaneously a running joke for massive worldwide publicity of fringe groups like the Raelians, and the core issue of an international movement at the United Nations in support of a treaty to ban the use of cloning techniques to produce a child (so called reproductive cloning). Yet, even though debates on human cloning have greatly increased since the birth of Dolly, the clone sheep, in 1997, we continue to wonder whether cloning is after all any different from other methods of medically assisted reproduction, and what exactly makes cloning an 'affront to the dignity of humans.' Categories we adopt matter mightily as they inform but can also misinform and lead to mistaken and unproductive decisions. And thus bioethicists have a responsibility to ensure that the proper categories are used in the cloning debates and denounce those who try to win the ethical debate through well-crafted labels rather than well-reasoned argumentations. But it is as important for bioethicists to take a position on broad issues such as human cloning and species altering interventions. One 'natural question' would be, for example, should there be an international treaty to ban human reproductive cloning?     

核移植与供体细胞

“多莉”羊的诞生是生物界的一个里程碑,它之所以引起如此大的轰动主要是因为它来源于培养的成年绵羊乳腺上皮细胞,这是人类第一次证明分化的体细胞可以被重编程后恢复全能性并最终分化发育成一个动物个体。这说明哺乳动物分化的体细胞核仍具有全套的遗传物质并能够被卵母细胞逆转恢复全能性。然而,关于多莉的供体细胞来源却一直是克隆领域的一个谜。由于体细胞克隆的效率非常低,而用于核移植的供体细胞悬液中往往含有多种类型的细胞,这使得我们很难确切地知道最终获得的克隆动物是来源于哪一种细胞。这种不确定性给我们研究核移植诱导体细胞重编程的机制带来了很大的困难,因此,对供体细胞的研究也是核移植研究领域的一个重要课题,这包括各种组织来源的体细胞是否均可以用于核移植,终末分化的体细胞是否能够用于核移植,组织干细胞是否更有利于体细胞重编程,供体细胞的分化状态是否与核移植的效率有关,死亡的体细胞是否也可以用于核移植等等。本文综述了核移植中与供体细胞相关的最新研究进展。     

Of totemism and ethnicity: Consciousness,practice and the signs of inequality*

In 1997, it was reported that a sheep had been successfully ‘cloned'at the Roslin Institute in Scotland. The sheep, named Dolly, attracted a great deal of attention and the idea of ‘cloning’ appeared to generate, in much of the western world at least, both anxiety and anticipation in equal measure. Discussion generated in the media immediately turned to the advantages and disadvantages of cloning people. I focus here on some of the responses to Dolly in the United States and in Britain. Drawing on fieldwork, pre‐Dolly, in the north of England, largue that the image of ‘the clone’ acts as a conceptual brake, as a marker of limits, to possibilities presented through new reproductive and genetic technologies. ‘Cloning’ provides an ethnographic window through which specific cultural understandings of kinship can be discerned.     

A reliable, noninvasive technique for spindle imaging and enucleation of mammalian oocytes

Factors affecting the efficiency of animal cloning remain to be elucidated. Enucleation of recipient oocytes is a critical step in cloning procedures and typically is performed by aspirating a portion of the cytoplasm underlying the first polar body. Enucleation is evaluated using epifluorescence after Hoechst staining for DNA, which may disrupt functions of the cytoplast, especially mitochondria. Mitochondrial DNA in Dolly and other cloned sheep has been shown to derive exclusively from recipient oocytes. Not only might evaluation of the aspirated karyoplast portion inadequately reflect the state of the cytoplast, it is also time consuming. Here we report a reliable, noninvasive technique for spindle imaging and enucleation of oocytes using a new microscope, the Pol-Scope. The efficiency of enucleation was 100%, and only 5.5% of the oocytes' mitochondria entered the karyoplast upon Pol-Scope-directed removal of the spindle. Moreover, Pol-Scope imaging of spindles and micromanipulation did not compromise the developmental competence of reconstituted oocytes and cytoplasts.     

Animal reproduction biotechnology in Poland

The key research areas of the Department are: in vitro production of embryos, embryo cryopreservation, animal transgenesis, cloning, cytometric semen sexing and evaluation. Research has been focused on the in vitro production of animal embryos, including the development of complex methods for oocyte maturation, fertilization and embryo culture. Moreover, experiments on long-term culturing of late preantral and early antral bovine ovarian follicles have been developed. Studies on the cloning of genetically modified pigs with "humanized" immunological systems have been undertaken. A cloned goat was produced from oocytes reconstructed with adult dermal fibroblast cells. The novel technique of rabbit chimeric cloning for the production of transgenic animals was applied; additionally, the recipient-donor-cell relationship in the preimplantation developmental competences of feline nuclear transfer embryos has been studied. Regarding transgenic animal projects, gene constructs containing growth hormone genes connected to the mMt promoter were used. Modifications of milk composition gene constructs with tissue-specific promoters were performed. Moreover, pigs for xenotransplantation and animal models of human vascular diseases have been produced. Over the last 15 years, our flow cytometry research group has focused its work on new methods for sperm quality assessment and sex regulation. In the 1970s, our team initiated studies on embryo cryopreservation. As a result of vitrification experiments, the world's first rabbits and sheep produced via the transfer of vitrified embryos were born.     

Animal breeding in the age of biotechnology: the investigative pathway behind the cloning of Dolly the sheep

History and Philosophy of the Life Sciences - This paper addresses the 1996 cloning of Dolly the sheep, locating it within a long-standing tradition of animal breeding research in Edinburgh. Far...     

克隆绵羊——多莉(DOLLY)的研究进展

多莉,第一例大型克隆哺乳动物,由一只称为FinnDorset的6岁母羊乳腺体细胞的基础生命物质经细胞核转移等操作而产生。成果公布后,持怀疑观点的学者提出,提供体细胞的母羊是否本身处在妊娠状态?推测多莉是由胚胎细胞产生,并非体细胞本身克隆所致。由于6岁的母羊死于1995年,只能用其冷冻保存的乳腺组织在Hannah研究中心进行细胞群体的分析。首先,用微卫星扩增技术,以三套引物进行测定;其次,进行DNA指纹分析来断定供体体细胞起源,以确定多莉的真实性。最后证实多莉来自成年母羊乳腺的体细胞。     

Cloned mice derived from somatic cell nuclei

In 1997, a cloned sheep "Dolly" was produced by nuclear transfer of somatic cell. The first birth of cloned mice derived from some somatic cells were succeeded in 1998. At present, it is shown that somatic cells, cumulus cells, fibroblasts and Sertoli cells can be used to the study of cloned animal as nuclear donor. In this study investigation was designed to compare with efficiency on the production of cloned embryos by using the microinjection and the electrofusion methods for nuclear transfer. Oocyte enucleation was performed with a micromanipulator. The oocyte was held by holding pipette, and was enucleated using a beveled pipette. Microinjection method: Cell's nucleus injection was carried out by piezo-micromanipulator. Cytochalasin B treated cumulus cell was aspirated into a injection pipette, and was broken its plasma membrane using the injection pipette. Then, the cumulus cell was injected into the enucleated ooplasm directly. Electrofusion method: The cell was aspirated into a beveled pipette, and then an aspirated cell was inserted into perivitelline space. Then, the pair of enucleated oocyte and cell was fused using electrical cell fusion apparatus. The reconstituted embryos were activated after nuclear transfer using St2+. Reconstituted embryos had been produced by the microinjection showed the embryonic development to over 8-cell stages. But, the rate of fragmentation of reconstituted embryos by the microinjection showed a little high rate in comparison with the electrofusion. When some reconstituted embryos by the microinjection were transplanted to pseudopregnant females' oviduct, 9 fetuses were observed at 14 days post coitum.     

Comparison of milk produced by cows cloned by nuclear transfer with milk from non-cloned cows

Cloning technologies, including embryo splitting and nuclear transfer, were introduced into dairy cattle breeding in the early 1980s. With the recent worldwide attention on the cloning of sheep ("Dolly") and cows ("Gene"), the potential food safety concerns for food products derived from cloned animals needs to be addressed. There has been no study of the composition of milk produced by cloned cows. In this preliminary study, we evaluated the composition of milk from 15 lactating non-embryonic cell cloned cows and six non-cloned lactating cows over a single season. The cloned cows came from five unique genetic lines and three distinct breeds. Milk samples were analyzed for total solids, fat, fatty acid profile, lactose, protein and compared to non-cloned and literature values. Gross chemical composition of milk from cloned cows was similar to that of the non-cloned cows and literature values. Our results lead us to conclude that there are no obvious differences in milk composition produced from cloned cows compared to non-cloned cows.     

Specific genetic modifications of domestic animals by gene targeting and animal cloning

The technology of gene targeting through homologous recombination has been extremely useful for elucidating gene functions in mice. The application of this technology was thought impossible in the large livestock species until the successful creation of the first mammalian clone "Dolly" the sheep. The combination of the technologies for gene targeting of somatic cells with those of animal cloning made it possible to introduce specific genetic mutations into domestic animals. In this review, the principles of gene targeting in somatic cells and the challenges of nuclear transfer using gene-targeted cells are discussed. The relevance of gene targeting in domestic animals for applications in bio-medicine and agriculture are also examined.     

Bioethical aspects of regenerative and reproductive medicine

The birth announced in 1997 of Dolly, the lamb cloned from the somatic mammary cells of an adult ewe, and the discovery of human embryonic stem cells in 1998 have been the most exciting developments in the biological sciences in the past decade. Reproductive somatic cell nuclear transfer (SCNT) in additional species has been inefficient in that relatively few births, harmful side effects and high fetal and neonatal death rates have resulted from many attempts. Ongoing debates about the ethics of reproductive SCNT have revealed that some researchers regard human reproductive SCNT as morally unacceptable in all circumstances, others see merit in reproductive SCNT in certain circumstances and others await more information before making judgment about the ethical status of the procedure. Regenerative medicine and emerging biotechnologies started to revolutionize the practice of medicine. Advances in stem cell biology, including embryonic and postnatal somatic stem cells, have made the prospect of tissue regeneration a potential reality. Mammal cloning experiments have provided new impetus to the prospect of regenerative medicine through stem cell research. The procedure of SCNT could be used to create the raw material to replace defective or senescent tissue as a natural extension of the biology of stem cells. Researchers working in reproductive medicine should consider the potential hope given to many patients against the requisite and ethically contentious creation of human blastocysts for therapeutic intent.     

Recent advancements in cloning by somatic cell nuclear transfer

Somatic cell nuclear transfer (SCNT) cloning is the sole reproductive engineering technology that endows the somatic cell genome with totipotency. Since the first report on the birth of a cloned sheep from adult somatic cells in 1997, many technical improvements in SCNT have been made by using different epigenetic approaches, including enhancement of the levels of histone acetylation in the chromatin of the reconstructed embryos. Although it will take a considerable time before we fully understand the nature of genomic programming and totipotency, we may expect that somatic cell cloning technology will soon become broadly applicable to practical purposes, including medicine, pharmaceutical manufacturing and agriculture. Here we review recent progress in somatic cell cloning, with a special emphasis on epigenetic studies using the laboratory mouse as a model.     

Remarkable differences in telomere lengths among cloned cattle derived from different cell types

Regarding cloned animals, interesting questions have been raised as to whether cloning restores cellular senescence undergone by their donor cells and how long cloned animals will be able to live. Focusing our attention on differences in telomere lengths depending on the tissue, we had produced 14 cloned cattle by using nuclei of donor cells derived from muscle, oviduct, mammary, and ear skin. Here, we show remarkable variation in telomere lengths among them using Southern blot analysis with telomere-specific probe. Telomere lengths in cloned cattle derived from muscle cells of an old bull were longer than those of a donor animal but were within the variation in normal calves. On the other hand, those derived from oviductal and mammary epithelial cells of an equally old cow were surprisingly shorter than any found in control cattle. The telomere lengths of cloned cattle derived from fibroblasts and oviductal epithelial cells of younger cattle showed the former and the latter results, respectively. In both cases, however, less telomere erosion or telomere extension from nuclear transfer to birth in most cloned cattle was observed in comparison with telomere erosion from fertilization to birth in control cattle. Embryonic cell-cloned cattle and their offspring calves were also shown to have telomeres longer than those in age-matched controls. These observations indicate that cloning does not necessarily restore the telomere clock but, rather, that nuclear transfer itself may commonly trigger an elongation of telomeres, probably more or less according to donor cell type. Remarkable variations among cloned cattle are suggested to be caused by variation in telomere length among donor cells and more or less elongation of telomere lengths induced by cloning.