[Progress on abnormal development of cloned pigs generated by somatic cell transfer nuclear].

Cloning, also known as somatic cell nuclear transfer (SCNT), is an asexual reproduction technique that reprograms differentiated cells to the totipotent state, and generates offspring with a genotype identical to the donor cells. Pig cloning technique holds great promise for propagating excellent breeding boars, generating genetically modified pigs, protecting rare and endangered pigs and studying the mechanisms of somatic cell nucleus reprogramming. However, cloned pigs suffer from various developmental defects, including low birth rate, low birth weight, and high stillbirth occurrence, neonatal mortality and congenital malformations, which severely hamper their applications. Errors in epigenetic reprogramming of donor nucleus are considered as the main causes of low cloning efficiency and abnormal embryonic development in cloned embryos and animals. However, most studies to correct the errors in epigenetic reprogramming of cloned pig embryos have not substantially improved the birth and survival rates of cloned pigs. In this review, we summarize the abnormal phenotypes, causes of abnormal development of cloned pigs and effective methods for improving pig cloning efficiency, thereby providing a reference for the future research to improve the development and survival rates of cloned pig embryos and cloned pigs.

Comprehensive evaluation of ubiquitous promoters suitable for the generation of transgenic cynomolgus monkeys†.

Nonhuman primates (NHPs) are considered to be the most valuable models for human transgenic (Tg) research into disease because human pathology is more closely recapitulated in NHPs than rodents. Previous studies have reported the generation of Tg NHPs that ubiquitously overexpress a transgene using various promoters, but it is not yet clear which promoter is most suitable for the generation of NHPs overexpressing a transgene ubiquitously and persistently in various tissues. To clarify this issue, we evaluated four putative ubiquitous promoters, cytomegalovirus (CMV) immediate-early enhancer and chicken beta-actin (CAG), elongation factor 1α (EF1α), ubiquitin C (UbC), and CMV, using an in vitro differentiation system of cynomolgus monkey embryonic stem cells (ESCs). While the EF1α promoter drove Tg expression more strongly than the other promoters in undifferentiated pluripotent ESCs, the CAG promoter was more effective in differentiated cells such as embryoid bodies and ESC-derived neurons. When the CAG and EF1α promoters were used to generate green fluorescent protein (GFP)-expressing Tg monkeys, the CAG promoter drove GFP expression in skin and hematopoietic tissues more strongly than in ΕF1α-GFP Tg monkeys. Notably, the EF1α promoter underwent more silencing in both ESCs and Tg monkeys. Thus, the CAG promoter appears to be the most suitable for ubiquitous and stable expression of transgenes in the differentiated tissues of Tg cynomolgus monkeys and appropriate for the establishment of human disease models.

The ultimate technology: the end of technology and the task of nature.

One of the most influential philosophers of the 20th century, Martin Heidegger (1889-1976), died prior to the remarkable cloning of the sheep Dolly and before Dr. Venter started his experiments on creating synthetic life, and he never explicitly discussed living technologies. However, by reinterpreting his notion of "modern technology," this article shows how it is possible to philosophically assess living technologies and to recognize ways in which Heidegger anticipated this phenomenon with his notion of cybernetics. The interpretation elucidates the fundamental process of technology becoming living and simultaneously presents living technology as the ultimate technology. The thesis of this article is that living technology is not just one more technology; rather, it is the perfection of technology as understood by Aristotle. Aristotle's thinking is in this way a key example of a profound reassessment of nature and technology. Aristotle clearly separates these two domains of being in his definition, but in doing so, he also connects them to one another in a highly influential way. Following this line of thought, the article finally offers an original perspective involving renewed respect for the perpetual self-unfolding nature of living technology.

A quantitative assessment of the risk of introducing foot and mouth disease virus into the United States via cloned bovine embryos.

The trade of livestock or their products between nations requires information on the risk of introducing infectious agents such as foot and mouth disease virus (FMDV). Although transmission pathways for FMDV vary, a recent concern in the United States (USA) is that it might enter via cloned embryos. A quantitative risk assessment model was developed to determine the scenarios (with mathematical probabilities) that could lead to the introduction and maintenance of FMDV via the importation of cloned bovine embryos. Using @RISK software with Monte Carlo simulation involving 50,000 iterations, the probability of introducing FMDV via cloned embryos was estimated to be 3.1 x 10(-7). Given the current cloning protocol, and assuming the annual importation of 250 to 1,700 (mean = 520) cloned embryos, the expected number of infected embryos ranges from 1.1 x 10(-7) to 4.4 x 10(-3) (mean = 1.6 x 10(-4)) per year. Critical pathway analysis showed that the risk of FMDV entering the USA by this route is extremely low.

Ethical aspects of tissue engineering: a review.

Tissue engineering (TE) is a promising new field of medical technology. However, like other new technologies, it is not free of ethical challenges. Identifying these ethical questions at an early stage is not only part of science's responsibility toward society, but also in the interest of the field itself. In this review, we map which ethical issues related to TE have already been documented in the scientific literature. The issues that turn out to dominate the debate are the use of human embryonic stem cells and therapeutic cloning. Nevertheless, a variety of other ethical aspects are mentioned, which relate to different phases in the development of the field. In addition, we discuss a number of ethical issues that have not yet been raised in the literature.

Regulating small things: genes, gametes and nanotechnology.

Biotechnology and nanotechnology both intersect with other technologies in ways that open new possibilities for further technological progress. The potential for increased convergence between technological fields highlights the need for regulatory frameworks to be integrated, flexible and responsive. Within a federal legal system such as Australia's, there is a need to ensure that we adopt a coordinated national approach to the crafting of regulatory solutions. In addition, there is a need for global cooperation in the development of international standards and regulatory harmonisation. Finally, this article considers the role that law plays in negotiating risk in relation to new technologies.

Piglets born from handmade cloning, an innovative cloning method without micromanipulation.

Porcine handmade cloning (HMC), a simplified alternative of micromanipulation based traditional cloning (TC) has been developed in multiple phases during the past years, but the final evidence of its biological value, births of piglets was missing. Here we report the first births of healthy piglets after transfer of blastocysts produced by HMC. As a cumulative effect of technical optimization, 64.3+/-2.3 (mean+/-S.E.M.) reconstructed embryos from 151.3+/-4.8 oocytes could be obtained after 3-4h manual work, including 1h pause between fusion and activation. About half (50.1+/-2.8%, n=16) of HMC reconstructed embryos developed to blastocysts with an average cell number of 77+/-3 (n=26) after 7 days in vitro culture (IVC). According to our knowledge, this is the highest in vitro developmental rate after porcine somatic cell nuclear transfer (SCNT). A total of 416 blastocysts from HMC, mixed with 150 blastocysts from TC using a cell line from a different breed were transferred surgically to nine synchronized recipients. Out of the four pregnancies (44.4%) two were lost, while two pregnancies went to term and litters of 3 and 10 piglets were delivered by Caesarean section, with live birth/transferred embryo efficiency of 17.2% (10/58) for HMC. Although more in vivo experiments are still needed to further stabilize the system, our data proves that porcine HMC may result in birth of healthy offspring. Future comparative examinations are required to prove the value of the new technique for large-scale application.

The emperor's new clones.

If regulators conclude that food from clones poses no more risk than food from other animals, the US and Europe could be on course for another biotech trade war.

Interspecies nuclear transfer: implications for embryonic stem cell biology.

Accessibility of human oocytes for research poses a serious ethical challenge to society. This fact categorically holds true when pursuing some of the most promising areas of research, such as somatic cell nuclear transfer and embryonic stem cell studies. One approach to overcoming this limitation is to use an oocyte from one species and a somatic cell from another. Recently, several attempts to capture the promises of this approach have met with varying success, ranging from establishing human embryonic stem cells to obtaining live offspring in animals. This review focuses on the challenges and opportunities presented by the formidable task of overcoming biological differences among species.

Somatic cell nuclei in cloning: strangers traveling in a foreign land.

The recent successes in producing cloned offspring by somatic cell nuclear transfer are nothing short of remarkable. This process requires the somatic cell chromatin to substitute functionally for both the egg and the sperm genomes, and indeed the processing of the transferred nuclei shares aspects in common with processing of both parental genomes in normal fertilized embryos. Recent studies have yielded new information about the degree to which this substitution is accomplished. Overall, it has become evident that multiple aspects of genome processing and function are aberrant, indicating that the somatic cell chromatin only infrequently manages the successful transition to a competent surrogate for gamete genomes. This review focuses on recent results revealing these limitations and how they might be overcome.

Cloning cattle: the methods in the madness.

Somatic cell nuclear transfer (SCNT) is much more widely and efficiently practiced in cattle than in any other species, making this arguably the most important mammal cloned to date. While the initial objective behind cattle cloning was commercially driven--in particular to multiply genetically superior animals with desired phenotypic traits and to produce genetically modified animals-researchers have now started to use bovine SCNT as a tool to address diverse questions in developmental and cell biology. In this paper, we review current cattle cloning methodologies and their potential technical or biological pitfalls at any step of the procedure. In doing so, we focus on one methodological parameter, namely donor cell selection. We emphasize the impact of epigenetic and genetic differences between embryonic, germ, and somatic donor cell types on cloning efficiency. Lastly, we discuss adult phenotypes and fitness of cloned cattle and their offspring and illustrate some of the more imminent commercial cattle cloning applications.

Developmental, behavioral, and physiological phenotype of cloned mice.

Cloning from adult somatic cells has been successful in at least ten species. Although generating viable cloned mammals from adult cells is technically feasible, prenatal and perinatal mortality is high and live cloned offspring have had health problems. This chapter summarizes the health consequences of cloning in mice and discusses possible mechanisms through which these conditions may arise. These studies have further significance as other assisted reproductive techniques (ART) also involve some of the same procedures used in cloning, and there are some reports that offspring generated by ART display aberrant phenotypes as well. At the moment, the long-term consequences of mammalian cloning remain poorly characterized. Data available thus far suggest that we should use this technology with great caution until numerous questions are addressed and answered.

Optimization of procedures for cloning by somatic cell nuclear transfer in mice.

Cloning by somatic cell nuclear transfer is a complex procedure that is dependent on correct interactions between oocyte and donor cell genome. These interactions require minimal insult to either the oocyte or the transplanted nucleus. Available data also indicate that reprogramming the donor cell genome may be slow, so that the cloned embryo expresses genes typical of the donor cell, and thus has different characteristics from normal embryos. Procedures that minimize damage to the donor genome and that address the unique characteristics of the cloned construct should enhance the efficacy of the method.

Handmade somatic cell cloning in cattle.

Apart from the biological and ethical problems, technical difficulties also hamper the improvement and widespread application of somatic cell nuclear transfer (NT). Recently introduced zona-free procedures may offer a solution for the latter problem. The most radical approach of these techniques is the so-called handmade cloning (HMC). It does not require micromanipulators because the manipulations required for both enucleation and nucleus transfer are performed by hand. The HMC technique includes manual bisection of zona-free oocytes, selection of cytoplasts by staining, and the simultaneous fusion of the somatic cell with two cytoplasts to produce a cloned embryo. HMC is a rapid and efficient technique that suits large-scale NT programs. It requires less expertise and time than traditional NT methods and the cost of equipment is significantly less. Production efficiency is high and embryo quality, in terms of pregnancy rates and live births, is not compromised. Although HMC has been developed particularly for bovine NT, the technique is applicable to other species. The method may become a useful tool for both experimental and commercial somatic cell cloning because it allows for standardization of procedures and provides the possibility of automation.

Are there any normal clones?

An exhaustive study of the fidelity of a clone to its parent is prohibitive because of cost and the necessary scope of experimental design. Therefore, these data must be gathered from existing observational evidence. This in itself cannot provide a definitive accounting of the abnormalities and variation found among clones or between clones and parents because there is no standardization in the data points collected between one study and another. This literature survey shows that clone developmental abnormalities, variation among clones, and variation between clone and parent are prevalent at most stages of development (cleavage, placental, fetal, neonatal, maturity), and that occasionally the observed variation greatly exceeds that which might be expected. Some variation can be explained by differences in protocols and procedures between studies. The choice of nuclear donor cell is particularly influential of variation observed between a clone and its parent. In general, however, it appears that there is an inherent stochastic response to nuclear transfer that results in clone infidelity and variation. The survey of characteristics of clone infidelity to parent and documentation of abnormalities provided here should not be viewed as exhaustive or limiting in the recording of such data from future studies. Because controlled hypothesis testing of clone fidelity or clone health may not be possible, meticulous documentation of such observational evidence is a valuable contribution to the field.

Molecular genetics: DNA analysis of a putative dog clone.

In August 2005, Lee et al. reported the first cloning of a domestic dog from adult somatic cells. This putative dog clone was the result of somatic-cell nuclear transfer from a fibroblast cell of a three-year-old male Afghan hound into a donor oocyte provided by a dog of mixed breed. In light of recent concerns regarding the creation of cloned human cell lines from the same institution, we have undertaken an independent test to determine the validity of the claims made by Lee et al..