Somatic Cell Nuclear Transfer

Somatic prison cell nuclear transfer (SCNT) was starting time proposed as a fashion to assess genetic totipotency of somatic cells within the context of whether genetic fabric was lost every bit cells differentiate.

From: Encyclopedia of Reproduction (Second Edition) , 2018

Cloning Pigs by Somatic Cell Nuclear Transfer

Kiho Lee , Randall S. Prather , in Principles of Cloning (Second Edition), 2014

Somatic prison cell nuclear transfer (SCNT) has expanded the utilization of pigs to various fields including biomedical inquiry. Even so, the efficiency of SCNT is poor, and thus practical awarding of SCNT is limited. Various approaches accept been attempted to increase the overall efficiency of SCNT. Improvements in in vitro civilisation of oocytes and embryos can help in production of high quality pig embryos. A wide molecular approach, such as next generation deep sequencing, has shown that it tin provide clues to understanding the physiology of embryos under in vitro civilisation and lead united states of america to develop a novel culture method. Unlike reagents have been used to assist reprogramming of donor cells during SCNT and to correct some abnormally expressed genes in SCNT embryos. Still, the search for an platonic donor cell type for efficient reprogramming remains an unfinished chore. These combined efforts provide a vivid prospective for SCNT in pigs.

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Nuclear Transfer for Stem Cells

Alan Trounson , in Principles of Cloning (Second Edition), 2014

Somatic cell nuclear transfer for pluripotent stalk cells is established for animal species, in detail in the mouse and non-human primates. Nevertheless, in that location take been major difficulties in deriving pluripotent stalk cells by nuclear transfer in the human being. Despite this difficulty, in that location are potent interests in establishing somatic cell nuclear transfer (SCNT) in the human for written report of early man evolution, product of gametes for infertile/sterile patients, comparisons of developmental and differentiation potential with induced pluripotential stem cells (iPSCs), and enquiry in stem prison cell biological science. The combination of oocyte derived factors and transcription factors for efficient and constructive reprogramming of somatic nuclei for pluripotent stem cells is a stiff inducement for farther inquiry in SCNT.

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Agricultural and Related Biotechnologies

Vilceu Bordignon , in Comprehensive Biotechnology (Tertiary Edition), 2019

iv.38.3.ane Cloning for Rescuing Endangered Species

SCNT cloning represents a method for preventing the extinction of endangered species. An important limitation for the cloning of endangered species is the availability of host oocytes from a related species. An culling to overcome this limitation is interspecies (cross-species) SCNT, which consists in producing cloned embryos using a nuclear donor cell and a host cytoplast from a different species. Although embryos have been generated not only from interspecies but also from intergeneric (using donor nuclei and recipient cytoplasts from unlike genera) SCNT in a number of species, only few alive cloned animals have been produced by interspecies SCNT until at present.

The cloning of the concluding surviving cow of the Enderby Island cattle brood using host oocytes from a different bovine breed represents the first successful application of SCNT for rescuing an endangered animal. 9 All the same, in that study donor nuclei, host oocytes, and the recipient females used for embryo transfer were all of the aforementioned species. Attempts for interspecies SCNT were initially made past transferring nuclei from sheep, pig, monkey, or rat cells into bovine cytoplasts, which resulted in embryo development, just at that place were no pregnancies established following embryo transfer to surrogate females. Other attempts for interspecies SCNT resulted in pregnancies, but not in survival to term, for example, after the transfer of embryos reconstructed with argali wild sheep (Ovis ammon) nuclei and domestic sheep (Ovis aries) oocytes. Examples of successful cloning through interspecies SCNT include Gaur (Bos gaurus) using domestic cattle (Bos taurus) oocytes; mouflon (Ovis orientalis musimon) using domestic sheep (Ovis aries) oocytes; African wild cat (Felis silvestris lybica) using domestic cat (Felis catus) oocytes; Gray wolf (Canis lupus) using domestic canis familiaris (Canis familiaris) oocytes; and The bucardo (Pyrenean ibex), an extinct wild goat (Capra pyrenaica pyrenaica), using domestic goat (Capra hircus) oocytes. This indicates that interspecies cloning can be used to rescue endangered species, at least when oocytes and surrogate females of closely related species are bachelor. x–12

Other exciting results regarding the potential use of SCNT for the revival of already extinct species were obtained by cloning mice from nuclei recovered from mice bodies that remained frozen for xvi   years. 13 These findings reopened the interest in the utilise of SCNT cloning to bring back extinct animals. For example, attempts to clone the woolly mammoth (Mammuthus primigenius) could be performed using Asian elephants (Elephas maximus) as oocyte donors and as surrogate mothers for embryo transfer.

A major obstacle in using SCNT to clone endangered animals is the species-specificity barrier for embryo transfer and the consistent unavailability of suitable foster mothers. And then far, there are no animals cloned using foster mothers and SCNT embryos of different genera. Interestingly, pregnancies were obtained in domestic cat females used as foster mothers for the transfer of SCNT embryos produced using nuclei from the behemothic pandas (Ailuropoda melanoleuca) or from domestic cats (F . catus) and oocytes from rabbits (Oryctolagus cuniculus). Although there were no pregnancies to term, an autopsy analysis performed 21   days after embryo transfer in a recipient female that received panda–rabbit and true cat–rabbit SCNT embryos revealed the presence of six fetuses, 4 derived from cat–rabbit and two from panda–rabbit embryos. 14

Given their central functions in the regulation of prison cell metabolism, programmed prison cell death, and prison cell signaling, mitochondria represent a potential effect affecting interspecies cloning because mitochondrial DNA heteroplasmy is produced in SCNT embryos, i.e., SCNT embryos normally possess mitochondrial DNA derived from both the nuclear donor prison cell and the host oocyte. This differs from normal fertilization where the sperm mitochondria are degraded and only those nowadays in the oocyte are maintained during embryo development, which results in mitochondrial homoplasmy. Although farther investigation is still necessary to determine the mitochondrial effect on development of interspecies SCNT embryos, it has been postulated that mitochondrial heteroplasmy may hamper development by creating incompatibility between the nucleus and the cytoplasm. Mitochondrial biogenesis, including replication and transcription, is regulated by nuclear-encoded proteins and may consequently be affected by the phylogenic distance between the nuclear donor cell and the host oocyte in SCNT embryos. Studies that assessed mitochondrial DNA heteroplasmy and segregation in SCNT embryos have in general reported a lower contribution of the nuclear donor prison cell compared to the host oocytes. However, additional studies are required to determine whether the degree of mitochondrial Dna heteroplasmy accounts for the evolution and viability of interspecies cloning. Among the possibilities to change the mitochondrial Deoxyribonucleic acid ratio in SCNT embryos, both the depletion and the increment of donor cell mitochondrial DNA accept been proposed. Although attempts for testing these approaches have been made, including the production of cloned animals homoplasmic for the mitochondrial DNA of the host oocytes, the consequences of changing the mitochondrial DNA ratio on the efficiency of interspecies cloning take non been sufficiently explored. 15,16

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Volume one

D.South. Koslov , A. Atala , in Encyclopedia of Biomedical Engineering, 2019

Somatic Jail cell Nuclear Transfer

Somatic cell nuclear transfer (SCNT) is the process of transplanting nuclei from adult cells into oocytes or blastocysts and allowing them to grow and differentiate, producing pluripotent cells. Figure one illustrates SCNT. This process has both reproductive and therapeutic implications. Both have the same initial process, removing an developed prison cell nucleus, placing into an oocyte, and stimulating information technology to abound with electricity or chemicals. This volition produce an embryo genetically identical to the donated prison cell nucleus: if planted in a uterus, a clone will develop. If the embryo is used for tissue development, it is considered therapeutic. The resultant cells are immunoidentical to the donor and capable of identical growth to a naturally formed embryo (Hochedlinger et al., 2002; Brambink et al., 2006). The interest of SCNT is to utilise the capacity to develop nonimmunogenic cells that tin develop into whatsoever tissue to replace the damaged organ.

Effigy 1. Somatic Cell Nuclear Transfer (SCNT) to produce replacement tissues using a patient's cells.

In SCNT, a feasible embryo is not always developed. A new study demonstrates that leaving the oocyte'due south genetic textile and merely adding the somatic cell genome will produce a triploid blastocyst that can lead to a feasible embryo that tin develop all three germ layers (Noggle et al., 2012). Several studies to engagement have demonstrated the capacity to remove jail cell nuclei from animal model cells, place in blastocysts and expand ex vivo, replace in the organism, and produce anatomic and functional renal units (Lanza et al., 2002; Atala et al., 1995; Yoo et al., 1996). Reproductive SCNT is banned in most countries due to ethical dilemmas, while therapeutic SCNT is an important form of enquiry.

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Characteristics and Characterization of Human Pluripotent Stem Cells

Anne Chiliad. Bang , Melissa K. Carpenter , in Essentials of Stem Cell Biology (Second Edition), 2009

Somatic Jail cell Nuclear Transfer

Somatic prison cell nuclear transfer (SCNT) takes advantage of a unique holding of the oocyte cytoplasm that allows somatic nuclei to exist reprogrammed to a pluripotent state. In this case, the nucleus of a somatic cell is transferred into an enucleated oocyte. The somatic nucleus is then reprogrammed, and fractional development to the ICM stage can occur in culture, followed past either transplantation into a prepared uterus in order to generate cloned animals, or harvesting the ICM to generate ESC lines (reviewed in Yang et al., 2007; Gurdon and Melton, 2008). In 2005, a group in South Korea reported the generation of homo ESCs from patient specific blastocysts created using SCNT. Unfortunately, this piece of work was later shown to be fraudulent, and to engagement, the generation of human being ESC lines using SCNT has non been reported.

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Embryology, Ethics of

S. Holm , in Encyclopedia of Applied Ideals (2nd Edition), 2012

Cloning past Somatic Cell Nuclear Transfer

SCNT is a technique in which a prison cell nucleus from a somatic jail cell is placed into an enucleated, unfertilized egg. This volition, in a small per centum of cases, atomic number 82 to a consummate reprogramming of the genetic material in the nucleus and enable the egg to start dividing and form an embryo. The resulting embryo tin can either exist used for research or the derivation of man embryonic stem cells or be brought to term. Using SCNT to produce embryonic stem cells is sometimes referred to every bit 'therapeutic cloning,' whereas the use of SCNT to produce live human beings is 'reproductive cloning.' Reproductive cloning has not been accomplished in humans, and it is unknown whether information technology has been tried, but it has been successful in a range of other mammals. Both the cosmos of stalk cells from embryos and the possibility of reproductive cloning heighten ethical issues that are covered in other articles.

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Genetically Tailored Hog Models for Translational Biomedical Enquiry

Bernhard Aigner , ... Eckhard Wolf , in Animal Models for the Study of Homo Affliction (2nd Edition), 2017

2.ii Somatic Jail cell Nuclear Transfer

Somatic cell nuclear transfer (SCNT, cloning) in pigs ( Betthauser et al., 2000; Onishi et al., 2000; Polejaeva et al., 2000) is carried out to produce mutations (knockout, -in) in divers loci of the porcine genome after using the sequence-specific nuclease-mediated techniques described above or after classical factor targeting by homologous recombination of targeting vector and host genome in the nuclear donor cells. In add-on, the method is also used for condiment gene transfer by random transgene integration in the nuclear donor cells. In both cases, preselection of donor cells with regard to the induced genetic modification, transgene expression and/or gender is possible. The production of genetically engineered animals by SCNT includes the transfection and selection of somatic donor cells in vitro, recovery and enucleation of recipient metaphase 2 oocytes, transfer of genetically modified somatic jail cell nuclei into the cytoplasm of the enucleated oocytes, activation of the reconstructed oocytes, and embryo transfer to recipients. In the instance that genetically modified donor cells are used, 100% genetically modified founder animals without genetic mosaicism are received. As prolonged in vitro civilization periods of the nuclear donor cells negatively influences the success of SCNT, use of pools of small clones of genetically modified nuclear donor cells rather than expansion of individual cell clones to high prison cell numbers may improve the efficiency of SCNT (Kurome et al., 2015).

The cloning efficiency is varying inside relatively depression values between 0.5% and v% offspring per transferred SCNT embryos. The successful embryonic, fetal, and neonatal evolution of the transferred embryos derived from SCNT depends on the correct epigenetic reprogramming of the donor cell nuclei. Insufficient epigenetic reprogramming may lead to an overall low cloning efficiency, too as to peri- and neonatal health problems of cloned mammals. Abnormal phenotypes of cloned pigs occur less oft than in other mammals, and they normally are not transmitted to the offspring of the affected clones (Cho et al., 2007; Shi et al., 2003; Suzuki et al., 2012). Genome-wide gene expression and DNA methylation profiling of different tissues in phenotypically normal cloned pigs and conventionally bred control pigs revealed differentially expressed genes and moderate alterations in DNA methylation (Gao et al., 2011; Park et al., 2011). Proteomic analyses of hearts of developed SCNT-derived Bama minipigs compared to controls too identified differentially expressed proteins thereby demonstrating that SCNT might effect in abnormal expression of important proteins in cardiac development (Shu-Shan et al., 2014). Thus, genetically engineered founders derived from SCNT may exist used in experiments only with caution with regard to the validity and reproducibility of the results.

Various somatic cell types were successfully used in the cloning procedure and numerous technical variations were established to increase the efficiency of porcine SCNT (Kurome et al., 2015). Furthermore, recombinant adeno-associated virus (rAAV) vectors (Rogers et al., 2008a), besides equally modified bacterial bogus chromosomes (Klymiuk et al., 2012a) were successfully used for efficient factor targeting in porcine cells. As minipigs are suitable experimental animals for biomedical inquiry, genetically modified cloned minipigs were produced by using minipigs every bit nuclear donors and common big-sized domestic subcontract breeds as both oocyte donors and recipients. This method combines the broad availability of domestic farm breeds with the biomedical value of minipigs (Estrada et al., 2008; Kurome et al., 2008).

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Interpreting the Stress Response of Early Mammalian Embryos and Their Stalk Cells

Y. Xie , ... D.A. Rappolee , in International Review of Cell and Molecular Biology, 2011

3.four Stress response in vitro during reprogramming of developed somatic nuclei to create pluripotent stem cells

Somatic cell nuclear transfer (SCNT) leads to stressed blastomeres in reprogramming embryos due to external stress of media mediated by suspected stress mechanisms. The fact that Oct4 office is similar Oct1 function in fibroblasts, in mediating preparation for and regulation of the ESC response to stress ( Kang et al., 2009a), is important for a number of reasons. Ane is that media stress during isolation of ESC from embryos derived from oocytes that underwent SCNT is compounded by the transition of the SCNT embryo from a stress response governed by the donor somatic nucleus to the reprogrammed nucleus which is presumably governed by a robust Oct4 response (Gao and Latham, 2004; Vassena et al., 2007), where it is also known that Oct4 can undergo positive feedback (Niwa et al., 2005). The combination of a nucleus that reprograms during a response to stressful and incompatible media with the presence of a stress response governed by Oct4 is likely to atomic number 82 to hypersensitive blastomeres that overreact to civilization stress and lower the efficacy of ESC production.

Oct4 mediates a stress response early during reprogramming after transfection of the "Yamanaka four" reprogramming transcription factors. In addition, during a 16-day written report of the ontogenesis of induced pluripotent stem (iPS) cells, the kickoff time point studied after reprogramming of an developed somatic cells using the "Yamanaka four" transcription factors was day 4 (Mikkelsen et al., 2008 and citations therein). At this time, the authors noted that a brisk stress kinome induction was detected past analysis of global mRNA microarray. Like the reprogramming of the donor nucleus during SCNT, difficulty in isolating iPS cells is likely to arise when culture stress is compounded by the overexpression of Oct4 which is needed to reprogram developed somatic nuclei. Overexpression of Oct4 that expresses a stress response domain makes the reprogramming cell hypersensitive to culture stress. Thus, derivation of ESC by SCNT and iPS cells by reprogramming may both benefit from characterizing and managing the stress response mediated past Oct4 and stress enzymes.

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Techniques for engineering float tissue

A. ATALA , in Biomaterials and Tissue Engineering in Urology, 2009

27.2.iii Therapeutic cloning (somatic cell nuclear transfer)

Somatic jail cell nuclear transfer (SCNT), or therapeutic cloning, entails the removal of an oocyte nucleus in culture, followed by its replacement with a nucleus derived from a somatic cell obtained from a patient. Activation with chemicals or electricity stimulates jail cell segmentation up to the blastocyst stage, and ES cells that are genetically identical to the patient tin be obtained from the newly generated inner cell mass.

At this point in our discussion, information technology is extremely of import to differentiate between the two types of cloning that exist – reproductive cloning and therapeutic cloning. Both involve the insertion of donor Deoxyribonucleic acid into an enucleated oocyte to generate an embryo that has identical genetic fabric to its DNA source. Yet, the similarities end in that location. In reproductive cloning, the embryo is and then implanted into the uterus of a pseudopregnant female to produce an infant that is a clone of the donor. A earth-famous example of this type of cloning resulted in the birth of a sheep named Dolly in 1997. 32 However, there are many ethical concerns surrounding such practices and, as a result, reproductive cloning has been banned in about countries.

While therapeutic cloning likewise produces an embryo that is genetically identical to the donor, this procedure is used to generate blastocysts that are explanted and grown in culture, rather than in utero. ES cell lines can so exist derived from these blastocysts, which are only allowed to grow upward to a 100-prison cell stage. At this time the inner jail cell mass is isolated and cultured, resulting in ES cells that are genetically identical to the patient. This process is detailed in Fig. 27.1. It has been shown that ES cells derived from the nuclei of fibroblasts, lymphocytes, and olfactory neurons are pluripotetent and can generate alive pups afterward injection into blastocysts. This shows that cells generated by SCNT take the aforementioned developmental potential equally blastocysts that are fertilized and produced naturally. 33–36 In addition, the ES cells generated by SCNT are perfectly matched to the patient'south immune system and no immunosuppressants would be required to prevent rejection should these cells be used in tissue engineering applications.

27.1. Strategies for therapeutic cloning and tissue engineering.

Although ES cells derived from SCNT incorporate the nuclear genome of the donor cells, mitochondrial Dna (mtDNA) contained in the oocyte could pb to immunogenicity after transplantation. In order to assess the histocompatibilty of tissue generated using SCNT, Lanza et al. 37 microinjected the nucleus of a bovine skin fibroblast into an enucleated oocyte. 37 Although the blastocyst was implanted (reproductive cloning), the purpose was to generate renal, cardiac, and skeletal musculus cells, which were then harvested, expanded in vitro, and seeded on to biodegradable scaffolds. These scaffolds were so implanted into the donor steer from which the cells were cloned to determine if the cells were histocompatible. Analysis revealed no evidence of a mounting T-jail cell response to the cloned renal cells, suggesting that rejection will non necessarily occur in the presence of oocytederived mtDNA. This finding represents a footstep forward in overcoming the histocompatibility problem of stalk cell therapy.

Although promising, SCNT has certain limitations that require further improvement before its clinical application, in addition to the upstanding considerations regarding the potential of SCNT-derived embryos to develop into offspring if implanted into a uterus. Commencement and foremost, this technique has not all the same been shown to work in humans. The initial failures and fraudulent reports of nuclear transfer in humans reduced excitement in the scientific customs and suggested that SCNT may not be platonic for homo applications, 38–40 although it was recently reported that not-human primate ES cell lines were generated by SCNT of nuclei from adult skin fibroblasts. 41, 42 In addition, before SCNT-derived ES cells can be used for any type of clinical therapy, careful assessment of the quality of the generated cell lines must exist made. For example, some cell lines generated by SCNT have independent chromosomal translocations and information technology is non known whether these abnormalities originated from the generation of aneuploid embryos, or if they occurred during ES cell isolation and civilization. The low 'success rate' of SNCT (only 0.7% of the nuclear transfers result in a viable blastocyst) and the inadequate supply of human oocytes further hinder the therapeutic potential of this technique. Still, these studies take renewed the promise that ethically neutral ES jail cell lines could one twenty-four hours be generated from homo cells to produce patient-specific stalk cells for utilise in tissue engineering and regenerative medicine applications.

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