Use Of Somatic Cell Nuclear Transfer Research Articles

Mouse somatic cell nuclear transfer: What has changed and remained unchanged in 25 years.

Somatic cell nuclear transfer (SCNT) is the only reproductive technology used to produce individuals from somatic cells by transferring them to enucleated oocytes. Although more than 25 years have passed since the first mammalian SCNT was reported in sheep, problems such as low birth rates and morphological abnormalities have persisted and limited its practical applications. The mouse is the ideal laboratory animal to unveil these questions due to its established reproductive technologies and extensive knowledge base of its genome and various strains. We investigated the causes of incomplete reprogramming after nuclear transfer of donor somatic cells and found that the loss of imprint in some placenta-specific imprinted genes could induce non-random SCNT abnormalities. By ameliorating aberrantly expressed imprinted genes, we succeeded in increasing the low birth rate and improving morphological abnormalities observed in SCNT fetuses. Furthermore, we sought appropriate mouse strains and cell types as nuclear donors to increase their developmental efficiencies and expand their applications in various fields. Peripheral blood cells are useful as ethical and economical cell species because they can be collected easily, even though SCNT embryos derived from hematopoietic cells show poor developmental abilities after reconstruction. Additionally, it is possible to obtain mice that are reactive to specific antigens of interest by using lymphocytes. Although there are still many limitations to the practical use of SCNT, its utilization is steadily expanding.

Oocytes obtained by ovum pick-up from live mares as an alternative to abattoir-derived oocytes for the development of equine embryos produced by somatic cell nuclear transfer

The production of equine embryos by somatic cell nuclear transfer (SCNT) is limited by the availability of immature oocytes. The main source of oocytes for SCNT comes from ovaries obtained from slaughter houses. However, in some countries there is no horse abattoirs or their number is limited which precludes the use of SCNT for the production of equine cloned embryos. Ovum pick-up (OPU) is a reproductive assisted technique which is growing rapidly in the equine industry which allows obtaining oocytes from live mares. OPU is mainly used in combination to ICSI (intracytoplasmic sperm injection) for the production of in vitro embryos. Oocytes collected from live recipient mares by OPU could be an alternative for SCNT in countries in which there are limited number of equine slaughter houses. However, it is unknown the effect of the oocytes’ source on the development of equine cloned embryos. The aim of this study was to compare the development of equine SCNT embryos produced using oocytes either retrieved by OPU or recovered from abattoir-sourced ovaries. For the study, a total of 1128 oocytes were used, of which 663 were from abattoir-sourced ovaries and 495 were from live mares programmed for OPU. The harvested ovaries were transported to the lab within 1 h for processing. The methods used for in vitro maturation (IVM), SCNT andembryo culture in vitro were identical for both sources of oocytes. The rates of maturation, cell fusion, cleavage and blastocyst formation at Day 7 were evaluated. Transferrable grade blastocysts were transferred to recipient mares 4 to 5 days after ovulation and pregnancy was diagnosed at Days 14 and 42 by ultrasound. The maturation rate of OPU-derived oocytes was lower than that of abattoir-derived oocytes (50.3 ± 2.7% vs . 61.9 ± 3.4%; P < 0.05). However, the rates of cell fusion (90.7 ± 2.6% vs. 81.9 ± 5.2%), cleavage (68.8 ± 3.9% vs . 61.9 ± 5.0%) and blastocyst formation (34.3 ± 2.8% vs. 25.4 ± 2.1%) were greater (P < 0.05) for OPU-derived embryos compared with abattoir-derived embryos. While similar proportions of OPU- and abattoir-derived blastocysts initiated pregnancy at Day 14, a greater proportion of OPU-derived blastocysts developed to Day 42 of gestation (13 of 50 embryos) compared with abattoir-derived blastocysts (3 of 27 embryos; P < 0.05). The results show that abattoir-sourced oocytes subjected to IVM have the potential to support the development of SCNT embryos that can initiate pregnancy. However, given the observed difference in subsequent in vivo development, the use of OPU-derived oocytes for equine SCNT embryo production is recommended. Further research is needed to improve the IVM of abattoir-sourced oocytes

Production of cloned cats using additional complimentary cytoplasm

Somatic cell nuclear transfer (SCNT) is an important technique for producing cloned animals. It, however, is inefficient when there is use of SCNT for cloned animal production. Cytoplasm injection cloning technology (CICT) was developed to overcome the inefficiencies of SCNT use of this purpose. The use of CICT involves additional cytoplasm fusing with enucleated oocytes to restore the cytoplasmic volume, thus improving the in vitro developmental competence and quality of cloned embryos. In this study, there was application of CICT in cats to improve the in vitro developmental competence of cloned embryos, as well as the production of the offspring. The results of this study were that fusion rate of the cloned embryos with use of the CICT method was greater than that with SCNT (80.0 ± 4.8% compared with 67.8 ± 11.3%, respectively), and more blastocysts developed with use of CICT than SCNT (20.0 ± 2.0% compared with 13.5 ± 5.0%, respectively). The 62 cloned embryos that were produced with use of CICT were transferred into five estrous synchronized recipients, and 151 cloned embryos produced using SCNT were transferred to 13 estrous-synchronized recipients. After the embryo transfer, there was birth from surrogate mothers of one live-born kitten that resulted using SCNT compared with three live-born kittens using CICT. The number of CICT-cloned embryos born was greater than that of SCNT-cloned embryos (4.8 ± 2.3% compared with 0.7 ± 1.3%, P < 0.05). These results indicate that the CICT technique can be used to produce cloned kittens, including endangered feline species.

Insights on bovine genetic engineering and cloning

Transgenic technology has become an essential tool for the development of animal biotechnologies, and animal cloning through somatic cell nuclear transfer (SCNT) enabled the generation of genetically modified animals utilizing previously modified and selected cell lineages as nuclei donors, assuring therefore the generation of homogeneous herds expressing the desired modification. The present study aimed to discuss the use of SCNT as an important methodology for the production of transgenic herds, and also some recent insights on genetic modification of nuclei donors and possible effects of gene induction of pluripotency on SCNT.

Cloning endangered felids using heterospecific donor oocytes and interspecies embryo transfer

Somatic cell nuclear transfer (SCNT) offers the possibility of preserving endangered species. It is one of the few technologies that avoids the loss of genetic variation and provides the prospect of species continuance, rather than extinction. Nonetheless, there has been a debate over the use of SCNT for preserving endangered species because of abnormal nuclear reprogramming, low efficiency and the involvement of extra mitochondrial DNA (mtDNA) of a different species in live offspring produced by interspecies SCNT. Despite these limitations, live endangered cloned animals have been produced. In the present paper, we describe recent research on the production of cloned embryos derived by fusion of wild felid fibroblast cells with heterospecific domestic cat cytoplasts and their viability after transfer into domestic cat recipients. In addition, we discuss epigenetic events that take place in donor cells and felid cloned embryos and mtDNA inheritance in wild felid clones and their offspring.

Stem cells, politics and the progress paradigm.

The analysis of Parliamentary debates provides the opportunity to assess the political context of Canadian legislation, particularly in controversial areas such as stem cell research. Parliamentary debates surrounding the recent Assisted Human Reproduction Act, (1) which lasted nearly a decade, were dominated by religious conservatives. At the forefront of the debate were issues such as the moral status of the embryo and the regulation of somatic cell nuclear transfer (SCNT). The resulting restrictive statutory provisions that ban SCNT ultimately arose from a convergence of rhetoric on dignity and the moral status of the embryo, and the resultant promotion of adult stem cell research. Approach We qualitatively analyzed the Canadian Hansard debates concerning stem cell policy from 1994 to 2004 by assigning codes to full text databases. (2) While the majority of the debates related to ethical issues, we focused on descriptions of scientific research by politicians, references to scientific progress, both proven and speculative, economic arguments, references to media coverage and direct quotes from scientific and other experts because the media and experts are major sources of information for politicians. The Parliamentary Debates Descriptions of Scientific Research We found Canadian politicians lack an understanding of scientific research, resulting in inflammatory statements on both costs and benefits of embryonic human stem cell research. Politicians generally misunderstood the process of obtaining embryonic stem cells and SCNT, a methodology used in both therapeutic and reproductive Both therapeutic and reproductive (human) cloning commence with the use of SCNT. However, reproductive cloning and therapeutic cloning for research purposes are used for different reasons with significantly different endpoints. Politicians generally conflate the two procedures. For instance, building upon the public's distaste for reproductive cloning, one member described the research of the Raelians, a Canadian research group who claim to have successfully cloned a human being, (3) in conjunction with therapeutic The member stated that we took one of [the Speaker's] cells, extracted the nucleus and put it into an ovum, one could stimulate it electrically and allow it to grow. The so-called therapeutic clone would be to take the immature model of Mr. Speaker and extract an organ, if he needed one, killing the clone in the That is so-called somatic nuclear cell transfer or therapeutic cloning. (4) Another member stated, I would suggest that most members of the House do not understand the difference between therapeutic and reproductive Certainly most people in Canada do not totally understand the difference between the two. In reality, there is not any difference. It is the same process. (5) Such statements demonstrate the lack of understanding of scientific research by politicians, and underscore the need for further education of all policy makers on issues relating to stem cell research prior to the creation of ideologically driven legislation. Alternatively, and of greater concern, these statements may also indicate a willingness of politicians to mislead the House and the public on controversial scientific research on the basis of ideology. Statements on Stem Cell Research The debates on both adult and embryonic stem cell research were dominated by the opposition parties, primarily the Canadian Alliance, which later merged with the Progressive Conservatives to become the Conservative Party, without any significant response from the governing party. It could be implied that the majority's silence demonstrated its unwillingness to assume the political risk of associating itself with the support of controversial research. Whatever the reason, however, the result was a debate largely centered on the moral status of the embryo and based upon the ideologies of the religious right and the god squad of the Liberal party. …

Endocrine Profiles and Growth Patterns of Cloned Goats

Two groups of goats produced by fetal somatic cell nuclear transfer (NT) were monitored to evaluate the similarities in growth patterns among cloned animals. Clone group I consisted of five Toggenburg females cloned from the same transgenic cell line and born to different recipient does. Clone group II consisted of two Saanen does born as twins to a single recipient female from a second transgenic cell line. Each cell line was constructed with a transgene (different for each clone group) that would express, producing a protein product in the milk during lactation of the does. Weight, hip height, and circulating levels of growth-related hormones were monitored at weekly and monthly intervals for comparison within the clone groups. A contemporary group (group III) consisting of seven crossbred does of similar ages and weights was also monitored for baseline endocrine values during the study. Serum samples from all groups were analyzed for growth hormone (GH), insulin-like growth factor I (IGF-I), triiodothyronine (T3), thyroxine (T4), and insulin via standard laboratory radioimmunoassay procedures. The averaged standard deviation from the mean was used to evaluate similarities within the groups of cloned does and the does in the contemporary group. The does in clone group II were less variable than the goats in clone group I for weight and hip height. This was perhaps due to a recipient effect. The two groups of cloned females were less variable than the contemporary group for circulating IGF-I and T4 concentrations. In contrast, the two groups of cloned does had at least one cloned group that was more variable than the contemporary group for GH, T3, and insulin. The animal variation, measured by the average standard deviation from the mean, of the cloned does is possibly due to environmental effects encountered in utero and/or in the postnatal period, as well as possible mitochondrial DNA differences between the cell line and donor oocytes used for NT. The variation among cloned does in this study indicates that the use of somatic cell NT to reproduce identical phenotypes may not succeed in all situations.

Teach the genie's master to be responsible.

Conventional wisdom says that you “can't put the genie back in the bottle,” but that's simply not true. And, when we think about it, do we really want to put the genie back in the bottle, or to better train and restrain its master? Take, for example, the atom and hydrogen bombs, the original genies. The devastation and destruction at Hiroshima and Nagasaki were unlike anything the world had or has since ever witnessed. Although the predominant thinking today is that the evil done by these bombs far outweighs the good, even though they brought a speedy resolution to World War II, it is arguable that the genie was responsible for more good than evil because there was more to the technology than just bombs. There was energy. Nuclear energy. A direct technological product of the escaped genie fuels power plants around the world. It also provides light and heat and increases the standard of living of hundreds of millions of people globally. What an advanced, civilized society must do with such dualistic technologies is not blindly try to stuff them back in the proverbial bottle, but rather, harness the technology for the good of society by providing and enforcing ethical and legal codes of conduct and use. And with many technologies, society has done so. If you still believe you can't put the genie back in the bottle, remember, there has never been another nuclear bomb dropped. Also, consider other potentially dual-use technologies such as guns, airplanes, and DNA typing. Society accepts that guns can morally and legally be used to defend one's life or the life of another, but they cannot be used to take an innocent life without significant consequences. Each day, airplanes increase understanding by bringing people and cultures closer together but, when hijacked and used as a missile, the world community condemns the actions and demands accountability. DNA technology has identified many murderers and rapists, but it can also be used to restrict people's liberty and invade their privacy. With the development and latest use of a nascent technology, society is once again confronted with the choice of abandoning a technology because of its potentially undesirable use or reigning in its power for the good of society. The technology of somatic cell nuclear transfer (SCNT), which famously produced Dolly the cloned sheep as well as clones in other species, has recently been used in humans but not for cloning. The technology was employed by a young Chinese couple to produce a genetically new individual. In other words, rather than use the nucleus of an already existing person or animal, as in Dolly's case, an embryo was grown in a laboratory, and the nucleus was removed and placed within an awaiting enucleated egg. SCNT technology was used in this case because, although the man's sperm was able to fertilize the woman's egg, a defective mechanism in the cytoplasm of the embryo produced by the couple inhibited the fertilized egg from developing properly. The woman became pregnant with triplets, which were selectively reduced to twins. The twins were born prematurely and died shortly after birth. As with the other technologies mentioned, the potential of SCNT technology is realized only at the hands of humankind. The use of SCNT to assist infertile women/couples pioneered by Dr James Grifo of New York University was, in Dr Grifo's opinion, overly burdened by US laws and regulations. Because of this restrictive environment, Dr Grifo had Dr Zhuang Guanglun of Sun Yat-Sen University perform the procedure in China. But herein lies the essence of harnessing the possibilities of great technologies—responsible use. It took 267 attempts before the successful birth of Dolly. Although initially seemingly healthy, she died young. Scientists speculate her death was due to expedited aging resulting from the origin of her birth. Furthermore, many animal “clones” successfully born using SCNT tend to be larger than average, yet it is not known why or if their birth technology plays a role. Similarly, it is not known what, if any, role the technology played in the premature and ultimate death of the human twins. It is easy to argue that such technology should never be used because of its potential for misuse in cloning humans and because the use of an untested, unperfected, unsafe technology in a nonemergency situation is irresponsible; however, it could also be argued that attempts to force the SCNT genie back into its bottle would be just as irresponsible. In an attempt to be a responsible master, the United States via the Food and Drug Administration created strict guidelines for SCNT research, but they were ignored, only to engender a reflexive all-out ban on the technology's use in China and other countries. Who's responsible now?

Regulation of assisted reproductive technologies in the United States

The regulation of any social activity is a complex activity, and perhaps this is no more so than in medicine where the interests of patients, profes sionals, society, and government can be in conflict. A special area of regula tory concern involves assisted reproductive technologies (ART), which are medical procedures involving the laboratory manipulation of gametes (sperm and eggs). In vitro fertilization (IVF) is the most commonly performed ART, whereas other technologies include intracytoplasmic sperm injection (ICSI), preimplantation genetic diagnosis/screening (PGD/S) and assisted hatching (AH). The use of donor eggs (oocytes) and surrogates (gestational carrier, host uterus) are also considered ART procedures. Donor insemina tion (DI) by itself is generally not considered an ART procedure. Somatic cell nuclear transfer (SCNT) or "cloning" has been classified into thera peutic cloning or the use of SCNT to produce cells and tissues that can be used to treat disease, and is considered appropriate by many people, and reproductive cloning, the use of SCNT to create a genetically "identical" individual, which is considered an inappropriate use of technology by almost everyone. ART technologies are also used to create stem cells for both research and potential therapeutic purposes. Why should these technologies be regulated, what should be regulated, how should it be done, and who should be responsible? These are difficult questions that have been answered differently in many countries and by