Talk:Somatic Cell Nuclear Transfer
Control of inner cells' proportion by asymmetric divisions and ensuing resilience of cloned rabbit embryos
Development. 2018 Mar 22. pii: dev.152041. doi: 10.1242/dev.152041. [Epub ahead of print]
Fabrèges D1, Daniel N2, Duranthon V2, Peyriéras N3.
Abstract Mammalian embryo cloning by nuclear transfer has a low success rate. This is hypothesized to correlate with a high variability of early developmental steps segregating outer cells, fated to extraembryonic tissues, from inner cells, giving rise to the embryo proper. Exploring the cell lineage of wild-type embryos (WT) and clones, imaged in toto until hatching, highlights the respective contributions of cell proliferation, death and asymmetric divisions to phenotypic variability. Preferential cell death of inner cells in clones, probably pertaining to the epigenetic plasticity of the transferred nucleus, is identified as a major difference with consequences on the inner cell proportion. In WT and clones, similar patterns of outer cell asymmetric divisions are shown to be essential to the robust inner cell proportion observed in WT. Asymmetric inner cell division, not described in mice, is identified as a regulator of the inner cell proportion, likely to give rise to resilient clones. KEYWORDS: 3D+time 2-photon imaging; Asymmetrical divisions; Cell death; Digital specimens; In silico experimentation; Rabbit preimplantation development; Somatic cell nuclear transfer; Spatial cell segregation PMID: 29567671 DOI: 10.1242/dev.152041
Healthy ageing of cloned sheep
Nat Commun. 2016 Jul 26;7:12359. doi: 10.1038/ncomms12359.
Sinclair KD1, Corr SA1, Gutierrez CG1,2, Fisher PA1, Lee JH1, Rathbone AJ1, Choi I1, Campbell KH1, Gardner DS1.
The health of cloned animals generated by somatic-cell nuclear transfer (SCNT) has been of concern since its inception; however, there are no detailed assessments of late-onset, non-communicable diseases. Here we report that SCNT has no obvious detrimental long-term health effects in a cohort of 13 cloned sheep. We perform musculoskeletal assessments, metabolic tests and blood pressure measurements in 13 aged (7-9 years old) cloned sheep, including four derived from the cell line that gave rise to Dolly. We also perform radiological examinations of all main joints, including the knees, the joint most affected by osteoarthritis in Dolly, and compare all health parameters to groups of 5-and 6-year-old sheep, and published reference ranges. Despite their advanced age, these clones are euglycaemic, insulin sensitive and normotensive. Importantly, we observe no clinical signs of degenerative joint disease apart from mild, or in one case moderate, osteoarthritis in some animals. Our study is the first to assess the long-term health outcomes of SCNT in large animals.
PMID 27459299 DOI: 10.1038/ncomms12359
The effect of amniotic membrane stem cells as donor nucleus on gene expression in reconstructed bovine oocytes
Int J Dev Biol. 2016;60(4-5-6):95-102.
Nazari H1, Shirazi A, Shams-Esfandabadi N, Afzali A, Ahmadi E.
Nuclear reprogramming of a differentiated cell in somatic cell nuclear transfer (SCNT) is a major concern in cloning procedures. Indeed, the nucleus of the donor cell often fails to express the genes which are a prerequisite for normal early embryo development. This study was aimed to evaluate the developmental competence and the expression pattern of some reprogramming related genes in bovine cloned embryos reconstructed with amniotic membrane stem cells (AMSCs) in comparison with those reconstructed with mesenchymal stem cells (MSCs) and adult fibroblasts (AF) as well as with in vitro fertilized (IVF) oocytes. In vitro matured abattoir-derived oocytes were considered as recipients and a hand-made cloning technique was employed for oocyte enucleation and nuclear transfer (NT) procedures. The expression pattern of genes involved in self-renewal and pluripotency (POU5F1, SOX2, NANOG), imprinting (IGF2, IGF2R), DNA methylation (DNMT1, DNMT3A), histone deacetylation (HDAC2), and apoptosis (BAX, BCL2) were evaluated in NT and IVF derived embryos. Despite the insignificant difference in cleavage rate between reconstructed and IVF oocytes, the blastocyst rate in the IVF group was higher than that of other groups. Among reconstructed oocytes, a higher blastocysts rate was observed in MSC-NT and AMSCs-NT derived embryos that were significantly higher than AF-NT derived ones. There were more similarities in the expression pattern of pluripotency and epigenetic modification genes between MSC-NT and IVF derived blastocysts compared with other groups. In conclusion, considering developmental competence, AMSCs, as alternative donors in SCNT procedure, like MSCs, were prone to have more advantage compared with AF. PMID 27389982
Effect of Acteoside as a Cell Protector to Produce a Cloned Dog
PLoS One. 2016 Jul 18;11(7):e0159330. doi: 10.1371/journal.pone.0159330. eCollection 2016.
Lee JH1, Chun JL1, Kim KJ1, Kim EY1, Kim DH1, Lee BM1, Han KW1, Park KS1, Lee KB2, Kim MK1.
Somatic cell nuclear transfer (SCNT) is a well-known laboratory technique. The principle of the SCNT involves the reprogramming a somatic nucleus by injecting a somatic cell into a recipient oocyte whose nucleus has been removed. Therefore, the nucleus donor cells are considered as a crucial factor in SCNT. Cell cycle synchronization of nucleus donor cells at G0/G1 stage can be induced by contact inhibition or serum starvation. In this study, acteoside, a phenylpropanoid glycoside compound, was investigated to determine whether it is applicable for inducing cell cycle synchronization, cytoprotection, and improving SCNT efficiency in canine fetal fibroblasts. Primary canine fetal fibroblasts were treated with acteoside (10, 30, 50 μM) for various time periods (24, 48 and 72 hours). Cell cycle synchronization at G0/G1 stage did not differ significantly with the method of induction: acteoside treatment, contact inhibition or serum starvation. However, of these three treatments, serum starvation resulted in significantly increased level of reactive oxygen species (ROS) (99.5 ± 0.3%) and apoptosis. The results also revealed that acteoside reduced ROS and apoptosis processes including necrosis in canine fetal fibroblasts, and improved the cell survival. Canine fetal fibroblasts treated with acteoside were successfully arrested at the G0/G1 stage. Moreover, the reconstructed embryos using nucleus donor cells treated with acteoside produced a healthy cloned dog, but not the embryos produced using nucleus donor cells subjected to contact inhibition. In conclusion, acteoside induced cell cycle synchronization of nucleus donor cells would be an alternative method to improve the efficiency of canine SCNT because of its cytoprotective effects. PMID 27428333
Artificial cloning of domestic animals
Proc Natl Acad Sci U S A. 2015 Jul 21;112(29):8874-8. doi: 10.1073/pnas.1501718112.
Domestic animals can be cloned using techniques such as embryo splitting and nuclear transfer to produce genetically identical individuals. Although embryo splitting is limited to the production of only a few identical individuals, nuclear transfer of donor nuclei into recipient oocytes, whose own nuclear DNA has been removed, can result in large numbers of identical individuals. Moreover, clones can be produced using donor cells from sterile animals, such as steers and geldings, and, unlike their genetic source, these clones are fertile. In reality, due to low efficiencies and the high costs of cloning domestic species, only a limited number of identical individuals are generally produced, and these clones are primarily used as breed stock. In addition to providing a means of rescuing and propagating valuable genetics, somatic cell nuclear transfer (SCNT) research has contributed knowledge that has led to the direct reprogramming of cells (e.g., to induce pluripotent stem cells) and a better understanding of epigenetic regulation during embryonic development. In this review, I provide a broad overview of the historical development of cloning in domestic animals, of its application to the propagation of livestock and transgenic animal production, and of its scientific promise for advancing basic research. KEYWORDS: SCNT; cloning; embryo; livestock; nuclear transfer
Lessons learned from cloning dogs
Reprod Domest Anim. 2012 Aug;47 Suppl 4:115-9. doi: 10.1111/j.1439-0531.2012.02064.x.
Kim M, Oh H, Kim G, Park J, Park E, Jang G, Ra J, Kang S, Lee B. Source Department of Theriogenology and Biotechnology, Seoul National University, Seoul, Korea Stem Cell Research Center, RNL BIO, Seoul, Korea.
The aim of this article is to review dog cloning research and to suggest its applications based on a discussion about the normality of cloned dogs. Somatic cell nuclear transfer was successfully used for production of viable cloned puppies despite limited understanding of in vitro dog embryo production. Cloned dogs have similar growth characteristics to those born from natural fertilization, with no evidence of serious adverse effects. The offspring of cloned dogs also have similar growth performance and health to those of naturally bred puppies. Therefore, cloning in domestic dogs can be applied as an assisted reproductive technique to conserve endangered species, to treat sterile canids or aged dogs, to improve reproductive performance of valuable individuals and to generate disease model animals. © 2012 Blackwell Verlag GmbH.
Segregation of donor cell mitochondrial DNA in gaur-bovine interspecies somatic cell nuclear transfer embryos, fetuses and an offspring
Mitochondrion. 2012 Jul 21;12(5):506-513. [Epub ahead of print]
Imsoonthornruksa S, Srirattana K, Phewsoi W, Tunwattana W, Parnpai R, Ketudat-Cairns M. Source Embryo Technology and Stem Cell Research Center, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
The fate of foreign mitochondrial DNA (mtDNA) following somatic cell nuclear transfer (SCNT) is still controversial. In this study, we examined the transmission of the heteroplasmic mtDNA of gaur donor cells and recipient bovine oocytes to an offspring and aborted and mummified fetuses at various levels during the development of gaur-bovine interspecies SCNT (iSCNT) embryos. High levels of the donor cell mtDNA were found in various tissue samples but they did not have any beneficial effect to the survival of iSCNT offspring. However, the factors on mtDNA inheritance are unique for each iSCNT experiment and depend on the recipient oocyte and donor cell used, which might play an important role in the efficiency of iSCNT. Copyright © 2012 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
Improved cloning efficiency and developmental potential in bovine somatic cell nuclear transfer with the oosight imaging system
Cell Reprogram. 2012 Aug;14(4):305-11. Epub 2012 Jul 20.
Kim EY, Park MJ, Park HY, Noh EJ, Noh EH, Park KS, Lee JB, Jeong CJ, Riu KZ, Park SP. Source 1 Mirae Biotech, Seoul 143-854, Korea. Abstract Abstract In somatic cell nuclear transfer (SCNT) procedures, exquisite enucleation of the recipient oocyte is critical to cloning efficiency. The purpose of this study was to compare the effects of two enucleation systems, Hoechst staining and UV irradiation (hereafter, irradiation group) and Oosight imaging (hereafter, Oosight group), on the in vitro production of bovine SCNT embryos. In the Oosight group, the apoptotic index (2.8±0.5 vs. 7.3±1.2) was lower, and the fusion rate (75.6% vs. 62.9%), cleavage rate (78.0% vs. 63.7%), blastocyst rate (40.2% vs. 29.2%), and total cell number (128.3±4.8 vs. 112.2±7.6) were higher than those in the irradiation group (all p<0.05). The overall efficiency after SCNT was twice as high in the Oosight group as that in the irradiation group (p<0.05). The relative mRNA expression levels of Oct4, Nanog, Interferon-tau, and Dnmt3A were higher and those of Caspase-3 and Hsp70 were lower in the Oosight group compared with the irradiation group (p<0.05). This is the first report to show the positive effect of the Oosight imaging system on molecular gene expression in the SCNT embryo. The Oosight imaging system may become the preferred choice for enucleation because it is less detrimental to the developmental potential of bovine SCNT embryos.
Inheritance of mitochondrial DNA in serially recloned pigs by somatic cell nuclear transfer (SCNT)
Biochem Biophys Res Commun. 2012 Aug 10;424(4):765-70. Epub 2012 Jul 15.
Do M, Jang WG, Hwang JH, Jang H, Kim EJ, Jeong EJ, Shim H, Hwang SS, Oh KB, Byun SJ, Kim JH, Lee JW. Source Regenerative Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305 806, Republic of Korea.
Somatic cell nuclear transfer (SCNT) has been established for the transmission of specific nuclear DNA. However, the fate of donor mitochondrial DNA (mtDNA) remains unclear. Here, we examined the fate of donor mtDNA in recloned pigs through third generations. Fibroblasts of recloned pigs were obtained from offspring of each generation produced by fusion of cultured fibroblasts from a Minnesota miniature pig (MMP) into enucleated oocytes of a Landrace pig. The D-loop regions from the mtDNA of donor and recipient differ at nucleotide sequence positions 16050 (A→T), 16062 (T→C), and 16135 (G→A). In order to determine the fate of donor mtDNA in recloned pigs, we analyzed the D-loop region of the donor's mtDNA by allele-specific PCR (AS-PCR) and real-time PCR. Donor mtDNA was successfully detected in all recloned offspring (F1, F2, and F3). These results indicate that heteroplasmy that originate from donor and recipient mtDNA is maintained in recloned pigs, resulting from SCNT, unlike natural reproduction. Copyright © 2012 Elsevier Inc. All rights reserved.
Uncoupled embryonic and extra-embryonic tissues compromise blastocyst development after somatic cell nuclear transfer
PLoS One. 2012;7(6):e38309. Epub 2012 Jun 6.
Degrelle SA, Jaffrezic F, Campion E, Lê Cao KA, Le Bourhis D, Richard C, Rodde N, Fleurot R, Everts RE, Lecardonnel J, Heyman Y, Vignon X, Yang X, Tian XC, Lewin HA, Renard JP, Hue I. Source INRA, UMR 1198 Biologie du Développement et Reproduction, Jouy-en-Josas, France.
Somatic cell nuclear transfer (SCNT) is the most efficient cell reprogramming technique available, especially when working with bovine species. Although SCNT blastocysts performed equally well or better than controls in the weeks following embryo transfer at Day 7, elongation and gastrulation defects were observed prior to implantation. To understand the developmental implications of embryonic/extra-embryonic interactions, the morphological and molecular features of elongating and gastrulating tissues were analysed. At Day 18, 30 SCNT conceptuses were compared to 20 controls (AI and IVP: 10 conceptuses each); one-half of the SCNT conceptuses appeared normal while the other half showed signs of atypical elongation and gastrulation. SCNT was also associated with a high incidence of discordance in embryonic and extra-embryonic patterns, as evidenced by morphological and molecular "uncoupling". Elongation appeared to be secondarily affected; only 3 of 30 conceptuses had abnormally elongated shapes and there were very few differences in gene expression when they were compared to the controls. However, some of these differences could be linked to defects in microvilli formation or extracellular matrix composition and could thus impact extra-embryonic functions. In contrast to elongation, gastrulation stages included embryonic defects that likely affected the hypoblast, the epiblast, or the early stages of their differentiation. When taking into account SCNT conceptus somatic origin, i.e. the reprogramming efficiency of each bovine ear fibroblast (Low: 0029, Med: 7711, High: 5538), we found that embryonic abnormalities or severe embryonic/extra-embryonic uncoupling were more tightly correlated to embryo loss at implantation than were elongation defects. Alternatively, extra-embryonic differences between SCNT and control conceptuses at Day 18 were related to molecular plasticity (high efficiency/high plasticity) and subsequent pregnancy loss. Finally, because it alters re-differentiation processes in vivo, SCNT reprogramming highlights temporally and spatially restricted interactions among cells and tissues in a unique way.
Recovery of cell nuclei from 15,000 years old mammoth tissues and its injection into mouse enucleated matured oocytes
Proc Jpn Acad Ser B Phys Biol Sci. 2009;85(7):240-7.
Kato H, Anzai M, Mitani T, Morita M, Nishiyama Y, Nakao A, Kondo K, Lazarev PA, Ohtani T, Shibata Y, Iritani A. Source Institute of Advanced Technology, Kinki University, Wakayama, Japan. email@example.com Abstract Here, we report the recovery of cell nuclei from 14,000-15,000 years old mammoth tissues and the injection of those nuclei into mouse enucleated matured oocytes by somatic cell nuclear transfer (SCNT). From both skin and muscle tissues, cell nucleus-like structures were successfully recovered. Those nuclei were then injected into enucleated oocytes and more than half of the oocytes were able to survive. Injected nuclei were not taken apart and remained its nuclear structure. Those oocytes did not show disappearance of nuclear membrane or premature chromosome condensation (PCC) at 1 hour after injection and did not form pronuclear-like structures at 7 hours after injection. As half of the oocytes injected with nuclei derived from frozen-thawed mouse bone marrow cells were able to form pronuclear-like structures, it might be possible to promote the cell cycle of nuclei from ancient animal tissues by suitable pre-treatment in SCNT. This is the first report of SCNT with nuclei derived from mammoth tissues.
Conditioning of karyoplasts for producing somatic nuclear transferred gonadal germ cells in domestic chickens
J Reprod Dev. 2008 Jun;54(3):221-4. Epub 2008 Mar 19.
Kohara Y, Minematsu T, Aikawa T, Kanai Y, Tajima A. Source Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Japan.
The objective of this study was to establish a protocol for generating karyoplasts that can be used to produce somatic nuclear transferred gonadal germ cells (snt-GGCs) in domestic chickens. Karyoplasts were produced by centrifuging cultured fibroblasts from 10-day-old chick embryos at 10,000 x g in the presence of 1.0 microg/ml cytochalasin B. The number of karyoplasts was significantly (P<0.05) higher and the diameters of the karyoplasts were significantly (P<0.05) smaller when fibroblasts were centrifuged for 60 min than for 10 or 30 min. It was possible to generate snt-GGCs by electrofusion of GGCs with karyoplasts produced from cryopreserved or serum-starved fibroblasts. These results indicate that karyoplasts generated from 10-day-old chick embryos can be used to produce snt-GGCs even after cryopreservation and serum starvation of the fibroblasts.
The US FDA and animal cloning: risk and regulatory approach
Theriogenology. 2007 Jan 1;67(1):198-206. Epub 2006 Oct 19.
Rudenko L, Matheson JC. Source Center for Veterinary Medicine, US Food and Drug Administration, Department of Health and Human Services, HFV-100, Rockville, MD 20855, USA. firstname.lastname@example.org
The Food and Drug Administration's (FDA's) Center for Veterinary Medicine issued a voluntary request to producers of livestock clones not to introduce food from clones or their progeny into commerce until the agency had assessed whether production of cattle, swine, sheep, or goats by somatic cell nuclear transfer (SCNT) posed any unique risks to the animal(s) involved in the process, humans, or other animals by consuming food from those animals, compared with any other assisted reproductive technology (ART) currently in use. Following a comprehensive review, no anomalies were observed in animals produced by cloning that have not also been observed in animals produced by other ARTs and natural mating. Further systematic review on the health of, and composition of meat and milk from, cattle, swine, and goat clones and the progeny of cattle and sheep did not result in the identification of any food-consumption hazards. The agency therefore concluded that food from cattle, swine, and goat clones was as safe to eat as food from animals of those species derived by conventional means. The agency also concluded that food from the progeny of the clone of any species normally consumed for food is as safe to eat as those animals. The article also describes the methodology used by the agency to analyze data and draw these conclusions, the plans the agency has proposed to manage any identified risks, and the risk communication approaches the agency has used.