Mitochondrial metabolism assessment of lycaon-dog fetuses in interspecies somatic cell nuclear transfer.

Many studies have reported that interspecies somatic cell nuclear transfer (iSCNT) is considered the prominent method in preserving endangered animals. However, the development rate of iSCNT embryos is low, and there are limited studies on the molecular mechanism of the iSCNT process. This study evaluated the developmental potential of interspecies lycaon (Lycaon pictus)-dog embryos and assessed the mitochondrial content and metabolism of the produced cloned lycaon-dog fetus. Of 678 collected oocytes, 516 were subjected to nuclear transfer, and 419 reconstructed embryos with male lycaon fibroblasts were transferred into 27 surrogates. Of 720 oocytes, 568 were subjected to nuclear transfer and 469 reconstructed embryos with female lycaon fibroblasts were transferred into 31 surrogates. Two recipients who received female reconstructed embryos were identified as pregnant at 30 days. However, fetal retardation with no cardiac activity was observed at 46 days. Microsatellite analysis confirmed that the cloned lycaon-dog fetus was genetically identical to the lycaon donor cell, whereas mitochondrial sequencing analysis revealed that oocyte donor dogs transmitted their mtDNA. We assessed the oxygen consumption rate and mitochondrial content of the aborted lycaon-dog fetus to shed some light on the aborted fetus's cellular metabolism. The oxygen consumption rates in the lycaon-dog fetal fibroblasts were lower than those in adult dog, lycaon and cloned dog fetal fibroblasts. Furthermore, lycaon-dog fetal fibroblasts showed decreased proportions of live and active mitochondria compared with other groups. Overall, we hypothesized that nuclear-mitochondrial incompatibility affects pyruvate metabolism and that these processes cause intrauterine fetal death.

Methods for Estimating the Probability of Clonality in Cell Line Development.

Mammalian cell banks for biopharmaceutical production are usually derived from a single progenitor cell. Different methods to estimate the probability that the cell banks are clonally derived, or the probability of clonality (PoC), associated with various cloning workflows have been reported previously. In this review, a systematic analysis and comparison of the methods used to calculate the PoC are provided. As the single cell deposition and high-resolution imaging technologies continue to advance and the cloning workflow evolves, an aligned understanding and best practice on estimating the PoC is necessary to compare different cloning workflows adopted across the biopharmaceutical industry and it will help to accelerate regulatory acceptance.

A matter of life and longer life.

While the major scientific discoveries that would extend the length and health of human lives are not yet here, the research that could create them is already underway. As prospects for a world in which extended and improved lives inches closer into reality, the discourse about what to consider as we move forward grows richer, with corporate executives, ideologues, scientists, theologians, ethicists, investigative journalists, and philosophers taking part in imagining and anticipating the rich array of humanity's possible futures. Drawing from in-depth interviews with key stakeholders (n = 22), we offer empirical insights into key values and beliefs animating the "longevity movement," including what constitutes an ideal human state, the imperative to intervene, and the role of individual liberty and concerns for equality. Emerging from these interviews are common concerns about reducing suffering, preserving diversity in visions of successful aging and how best to promote access to a future that may not remain hypothetical for long.

Non-invasive assessment of culture media from goat cloned embryos associated with subjective morphology by gas chromatography - mass spectroscopy-based metabolomic analysis.

Pre-implantation embryo metabolism demonstrates distinctive characteristics associated with the development potential of embryos. We aim to determine if metabolic differences correlate with embryo morphology. In this study, gas chromatography - mass spectroscopy (GC-MS)-based metabolomics was used to assess the culture media of goat cloned embryos collected from high-quality (HQ) and low-quality (LQ) groups based on morphology. Expression levels of amino acid transport genes were further examined by quantitative real-time PCR. Results showed that the HQ group presented higher percentages of blastocysts compared with the LQ counterparts (P < 0.05). Metabolic differences were also present between HQ and LQ groups. The culture media of the HQ group showed lower levels of valin, lysine, glutamine, mannose and acetol, and higher levels of glucose, phytosphingosine and phosphate than those of the LQ group. Additionally, expression levels of amino acid transport genes SLC1A5 and SLC3A2 were significantly lower in the HQ group than the LQ group (P < 0.05, respectively). To our knowledge, this is the first report which uses GC-MS to detect metabolic differences in goat cloned embryo culture media. The biochemical profiles may help to select the most in vitro viable embryos.

Radiographic assessment of the skeletons of Dolly and other clones finds no abnormal osteoarthritis.

Our recent report detailing the health status of cloned sheep concluded that the animals had aged normally. This is in stark contrast to reports on Dolly (first animal cloned from adult cells) whose diagnoses of osteoarthritis (OA) at 5½ years of age led to considerable scientific concern and media debate over the possibility of early-onset age-related diseases in cloned animals. Our study included four 8-year old ewes derived from the cell line that gave rise to Dolly, yet none of our aged sheep showed clinical signs of OA, and they had radiographic evidence of only mild or, in one case, moderate OA. Given that the only formal record of OA in Dolly is a brief mention of a single joint in a conference abstract, this led us to question whether the original concerns about Dolly's OA were justified. As none of the original clinical or radiographic records were preserved, we undertook radiographic examination of the skeletons of Dolly and her contemporary clones. We report a prevalence and distribution of radiographic-OA similar to that observed in naturally conceived sheep, and our healthy aged cloned sheep. We conclude that the original concerns that cloning had caused early-onset OA in Dolly were unfounded.

[Cloning and law in Hungary]. / Klónozás és jog Magyarországon.

Reproductive human cloning is prohibited in Hungary, as in many other countries. Therapeutic human cloning is not prohibited, just like in many other countries. Stem cell therapy is also allowed. Article III, paragraph (3) of the Hungarian basic law (constitution) strictly forbids total human cloning. Article 1 of the Additional Protocol to the Oviedo Convention, on the Prohibition of Cloning Human Beings (1998) stipulates that any intervention seeking to create a human being genetically identical to another human being, whether living or dead, is prohibited. In Hungary, according to Article 174 of the Criminal Code, total human cloning constitutes a crime. Article 180, paragraph (3) of the Hungarian Act on Health declares that embryos shall not be brought about for research purposes; research shall be conducted only on embryos brought about for reproductive purposes when this is authorized by the persons entitled to decide upon its disposal, or when the embryo is damaged. Article 180, paragraph (5) of the Hungarian Act on Health stipulates that multiple individuals who genetically conform to one another shall not be brought about. According to Article 181, paragraph (1) of the Hungarian Act on Health, an embryo used for research shall be kept alive for not longer than 14 days, not counting the time it was frozen for storage and the time period of research.

Medical students and controversial ethical issues: results from the multicenter study SBRAME.

BACKGROUND: Medical students(MS) will face ethical issues throughout their lives as doctors. The present study aims to investigate medical students' opinions on controversial ethical issues and factors associated with these opinions. METHODS: SBRAME (Spirituality and Brazilian Medical Education) is a multicenter study involving 12 Brazilian medical schools with 5950 MS. Participants completed a questionnaire that collected information on socio-demographic data, medical schools characteristics, religious beliefs and opinions on controversial ethical issues. Of all MS, 3630 participated in the survey (61.0%). RESULTS: The sample was 53.8% women and the mean age was 22.5 years. In general, most MS have no objections to prescription of birth control (90.8%), adult stem cell use (87.5%), embryonic stem cell use (82.0%) and abortion for genetic reasons (51.2%). Approximately half of students have no objections to human cloning (47.3%), 45.7% to withdrawal of artificial life support, 41.4% to euthanasia and 23.3% to abortion for failed contraception. Socio-demographic data such as age, gender and income had little influence on MS opinions. On the other hand, medical schools characteristics (number of medical students in the university, year of medical school foundation, location of the university and type of university) and religious aspects (religious affiliation, religious attendance, non-organizational religiousness and intrinsic religiousness) were highly correlated with their opinions. In general, MS with more supportive opinions on controversial ethical issues were less religious and from non-traditional (newer), urban, public and bigger universities. CONCLUSION: The current study reveals MS have different opinions regarding controversial ethical issues. Noteworthy, these opinions seem to be shaped more by university characteristics and religious beliefs than socio-demographic data.

Un futuro no muy lejano. Gattaca revisitada / «The not-too-distant-future...¼ Gattaca revisited

La indicación temporal («un futuro no muy lejano¼) con la que comienza el film Gattaca (Andrew Niccol, 1997) lleva a reflexionar sobre las cuestiones éticas y legales que abre el campo de la reprogenética. Esa reflexión no puede postergarse. La misma implica considerar la historia del pensamiento y las prácticas eugenésicas y de su inscripción en el desarrollo de la ciencia, enmarcada a su vez en el capitalismo como modo de lazo social. Ello supone interrogar la lógica instrumental del capitalismo, y el margen que el sujeto y lo real representan con respecto a la misma, así como la relación entre la ley jurídica, el desarrollo tecnológico y el campo del sujeto. Se plantea, por último, el valor que tendría la reflexión ética para incidir en el desarrollo de las biotecnologías, y en particular de la reprogenética, así como en su regulación legal

The time indication («the not-too-distant-future¼) at the beginning of the film Gattaca (Andrew Niccol, 1997) leads us to reflect on the ethical and legal issues that the field of reprogenetics opens. That reflection cannot be postponed. It involves considering the history of eugenic thinking and practices within the development of science. Such development, in turn, took place in a context given by capitalism as a form of social tie. This implies questioning the instrumental logic of capitalism, and the margin that the subject and the real introduce, as well as the relationship between juridical law, biotechnological development (especially that concerning reprogenetics) and the subject. There is, finally, the question of what value ethical reflection could have to influence the development of biotechnology (especially reprogenetics) and its legal regulation

What justifies the United States ban on federal funding for nonreproductive cloning?

This paper explores how current United States policies for funding nonreproductive cloning are justified and argues against that justification. I show that a common conceptual framework underlies the national prohibition on the use of public funds for cloning research, which I call the simple argument. This argument rests on two premises: that research harming human embryos is unethical and that embryos produced via fertilization are identical to those produced via cloning. In response to the simple argument, I challenge the latter premise. I demonstrate there are important ontological differences between human embryos (produced via fertilization) and clone embryos (produced via cloning). After considering the implications my argument has for the morality of publicly funding cloning for potential therapeutic purposes and potential responses to my position, I conclude that such funding is not only ethically permissible, but also humane national policy.

[The embryonic stem cells research. Example of biotechnology progress under extra-scientific pressure]. / La Investigación con Células Troncales Embrionarias. Ejemplo de Progreso Biotecnológico bajo Presiones Extracientíficas.

The possibility to isolate, cultivate, preserve, characterize and differentiate Human Embryonic Stem Cells (ES) discovered by James Thomson and his colleagues in 1998 was a milestone in the history of Stem Cell Research. Immediately after this discovery many speculations were made about the therapeutic possibilities of ES, motivated by ideological, political and economic aspects. The episode made clear the lack of scientific rationality and ethics when assessing realities as meaningful as those of human embryos obtained by in vitro fertilization techniques (IVF) or human eggs. Therapeutic Cloning as a promise to produce ″tailored″ Stem Cells reported by Hwang and his team in 2004, ended up being a scandal within the scientific community. The technical difficulties and ethical controversies that arose from obtaining ES were insurmountable. In 2010 only two clinical trials were reported using these cells. Those trials were abandoned in late 2011 arguing financial reasons. The discovery of Induced Pluripotent Stem Cell (iPS) in 2006 in mice and in 2007 in humans, represented the possibility of obtaining pluripotent stem cells without the need to destroy embryos. Today, the absence of clinical trials using ES, caused by financial difficulties as a result of its ineffectiveness, anticipates that the use of ES will be limited to certain experimental controls. Probably, the main contribution of Embryonic Stem Cells will be the understanding that biomedical research should follow an ethically and rationally based rigorous method that cannot be ignore.

Assessment of MDA efficiency for genotyping using cloned embryo biopsies.

The possibility to genotype embryos prior to implantation would have advantages for increasing the speed of selection of cattle. Reliable genotyping requires more DNA than can be obtained from biopsies of embryos, if they are to remain viable. Multiple displacement amplification (MDA) is a whole genome amplification technique used to increase the amount of DNA from biopsies for analysis. Reduced genome coverage resulting in Allele Drop Out (ADO) at heterozygous loci or missing genotypes are drawbacks of MDA. The present article describes the correlation between the input DNA quantity or embryo biopsy size and MDA success. Missing genotypes and ADO drastically increased when fewer than 30-40 cells or the genomic equivalents were used. However, embryo viability was found to be reduced if biopsied with more than 10 cells. Therefore, in vitro cell culture was investigated as a means to increase the number of cells available and the genotyping reliability.

Ethical modernization: research misconduct and research ethics reforms in Korea following the Hwang affair.

The Hwang affair, a dramatic and far reaching instance of scientific fraud, shocked the world. This collective national failure prompted various organizations in Korea, including universities, regulatory agencies, and research associations, to engage in self-criticism and research ethics reforms. This paper aims, first, to document and review research misconduct perpetrated by Hwang and members of his research team, with particular attention to the agencies that failed to regulate and then supervise Hwang's research. The paper then examines the research ethics reforms introduced in the wake of this international scandal. After reviewing American and European research governance structures and policies, policy makers developed a mixed model mindful of its Korean context. The third part of the paper examines how research ethics reform is proactive (a response to shocking scientific misconduct and ensuing external criticism from the press and society) as well as reactive (identification of and adherence to national or international ethics standards). The last part deals with Korean society's response to the Hwang affair, which had the effect of a moral atomic bomb and has led to broad ethical reform in Korean society. We conceptualize this change as ethical modernization, through which the Korean public corrects the failures of a growth-oriented economic model for social progress, and attempts to create a more trustworthy and ethical society.

Comprehensive assessment of milk composition in transgenic cloned cattle.

The development of transgenic cloned animals offers new opportunities for agriculture, biomedicine and environmental science. Expressing recombinant proteins in dairy animals to alter their milk composition is considered beneficial for human health. However, relatively little is known about the expression profile of the proteins in milk derived from transgenic cloned animals. In this study, we compared the proteome and nutrient composition of the colostrum and mature milk from three lines of transgenic cloned (TC) cattle that specifically express human α-lactalbumin (TC-LA), lactoferrin (TC-LF) or lysozyme (TC-LZ) in the mammary gland with those from cloned non-transgenic (C) and conventionally bred normal animals (N). Protein expression profile identification was performed, 37 proteins were specifically expressed in the TC animals and 70 protein spots that were classified as 22 proteins with significantly altered expression levels in the TC and C groups compared to N group. Assessment of the relationship of the transgene effect and normal variability in the milk protein profiles in each group indicated that the variation in the endogenous protein profiles of the three TC groups was within the limit of natural variability. More than 50 parameters for the colostrum and mature milk were compared between each TC group and the N controls. The data revealed essentially similar profiles for all groups. This comprehensive study demonstrated that in TC cattle the mean values for the measured milk parameters were all within the normal range, suggesting that the expression of a transgene does not affect the composition of milk.

Assessment of reproduction and growth performance of offspring derived from somatic cell cloned pigs.

Since cloned pig was successfully produced, a new opportunity for porcine breeding industry to conserve genetic resources has been opened. However, there has been no report to investigate whether both somatic cell nuclear transfer (SCNT) pigs and their offspring have the characteristics of the donor breed. In this study, we compared the reproductive and growth performance of American Large White boars cloned by SCNT with the donor boar, and analyzed the test parameters, including semen quality, re-service rate, rate of parturition, and average daily gain. The results showed that these cloned boars and the donor boar had no significant differences in the tests (P > 0.05) and the growth performance of their offspring was similar to the naturally bred American Large White pigs. In summary, the reproductive and growth performance of cloned pigs are similar to the donor pig and within the normal range. This suggests that pigs cloned by SCNT have the potential to be used in reproduction and breeding.