Somatic Cell Nuclear Transfer

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Introduction

Dolly the Sheep
Dolly's sisters[1]
Mice cloned from adult keratinocytes[2]

In 1996 Dolly the sheep was the first animal to be produced by somatic cell nuclear transfer (SCNT) using an adult-derived somatic cell as nuclear donor. A somatic cell refers to the fact that a cell that is not a germ cell (spermatozoa, oocyte) is used to generate a zygote from which the embryo develops. This topic is closely related to Stem Cells.

A range of different cell types has now been successfully applied to a range of species (cattle, mice, goats, pigs, cats, rabbits, horses, rats, dogs and ferrets. (see review[3])

SCNT Links: Introduction | Stem Cells - Induced | Stem Cells - SCNT | Epigenetics | Stem Cells | ART | Fertilization | Week 1 | Category:Zygote

Some Recent Findings

  • Healthy ageing of cloned sheep[1] "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." Sheep Development
  • Review - Artificial cloning of domestic animals[4] "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."
  • Inheritance of mitochondrial DNA in serially recloned pigs by somatic cell nuclear transfer (SCNT)[5] "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. ... These results indicate that heteroplasmy that originate from donor and recipient mtDNA is maintained in recloned pigs, resulting from SCNT, unlike natural reproduction."
  • Number of blastomeres and distribution of microvilli in cloned mouse embryos during compaction[6] "We concluded that: (i) the cleavage of blastomeres in cloned embryos was slow at least before compaction; (ii) the distribution of microvilli in cloned, normal, parthenogenetic, and tetraploid embryos was coherent before and after compaction; and (iii) the initiation of compaction in somatic cell nuclear transfer (SCNT) embryos was delayed compared with that of intracytoplasmic sperm injection (ICSI) embryos."
More recent papers
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This table shows an automated computer PubMed search using the listed sub-heading term.

  • Therefore the list of references do not reflect any editorial selection of material based on content or relevance.
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Search term: Somatic Cell Nuclear Transfer

Jun-Xue Jin, Sanghoon Lee, Erif Maha Nugraha Setyawan, Anukul Taweechaipaisankul, Geon A Kim, Ho Jae Han, Curie Ahn, Byeong Chun Lee A potential role of knockout serum replacement as a porcine follicular fluid substitute for in vitro maturation: Lipid metabolism approach. J. Cell. Physiol.: 2018; PubMed 29345310

Huaqiang Yang, Jian Zhang, Xianwei Zhang, Junsong Shi, Yongfei Pan, Rong Zhou, Guoling Li, Zicong Li, Gengyuan Cai, Zhenfang Wu CD163 knockout pigs are fully resistant to highly pathogenic porcine reproductive and respiratory syndrome virus. Antiviral Res.: 2018; PubMed 29337166

Degong Ruan, Jiangyun Peng, Xiaoshan Wang, Zhen Ouyang, Qingjian Zou, Yi Yang, Fangbing Chen, Weikai Ge, Han Wu, Zhaoming Liu, Yu Zhao, Bentian Zhao, Quanjun Zhang, Chengdan Lai, Nana Fan, Zhiwei Zhou, Qishuai Liu, Nan Li, Qin Jin, Hui Shi, Jingke Xie, Hong Song, Xiaoyu Yang, Jiekai Chen, Kepin Wang, Xiaoping Li, Liangxue Lai XIST Derepression in Active X Chromosome Hinders Pig Somatic Cell Nuclear Transfer. Stem Cell Reports: 2018; PubMed 29337117

Jason A Koroghli, Elizabeth Floyd, Misha Regouski, Kerry Rood, Kirsten Gash, Kip Panter, Rusty Stott, Christopher J Davies, Irina A Polejaeva, Heloisa M Rutigliano Gene expression and lymphocyte population at the fetal-maternal interface in sheep pregnancies established by somatic cell nuclear transfer. Reprod. Fertil. Dev.: 2018; PubMed 29332623

Kyeoung-Hwa Kim, Eun-Young Kim, Su-Yeon Lee, Jung-Jae Ko, Kyung-Ah Lee Oocyte Cytoplasmic Gas6 and Heparan Sulfate (HS) are Required to Establish the Open Chromatin State in Nuclei During Remodeling and Reprogramming. Cell. Physiol. Biochem.: 2017, 45(1);37-53 PubMed 29316553

SCNT Animal Timeline


Cloned dog.jpg

Cloned Dog (2016)[12]

Process

  1. Somatic nucleus cell source - culture of somatic cells from nucleus donor.
  2. Oocyte - nucleus and the polar body are removed from oocyte by aspiration giving an enucleated oocyte.
  3. Injection - of a somatic cell between the zona pellucida and the membrane of the enucleated oocyte.
  4. Electrofusion - Introduction of the somatic cell nucleus (and cytoplasm) into the oocyte cytoplasm.
  5. Embryo clone - formed by an oocyte cytoplasm and a somatic cell nucleus containing two copies of chromosomes.
  6. Embryo transfer - into a surrogate dam generating clone (F0) with coat colour similar to that of the nucleus source.
  7. Clone offspring - (F1) generated by the sexual reproduction of the clone (F0) with a normal partner.

Oocyte Enucleation

Sheep OOcyte Nucleus[13]

Before a somatic cell nuclei can be introduced into an oocyte, the oocyte's own nucleus needs to be removed. This process of oocyte nuclear removal is described as "oocyte enucleation". A recent study in cattle found oocyte imaging had a higher efficiency. [14]

The oocyte nucleus can be identified by:

  1. Hoechst staining and UV irradiation.
  2. Oocyte imaging.

Somatic Cell Source

A number of different tissues have been used as the somatic cell nucleus source including:

  • ovarian cumulus cells
  • fibroblasts
  • mammary epithelium
  • lymphocytes
  • neural stem cells
  • olfactory
  • myoblasts

Epigenetics

Epigenetic profiles of SCNT bovine embryos[15]

Several studies have reported that introduction of the somatic nuclei leads to deleterious epigenetic changes including DNA methylation and histone acetylation.[16][17][18]

Mitochondria

Typically mitochondria are maternally inherited and any paternal (spermatozoa) mitochondria either do not enter the oocyte or are destroyed. A recent SCNT study in pig[5] has demonstrated a mixed inheritance pattern.

Inheritance of mitochondrial DNA in serially recloned pigs by somatic cell nuclear transfer (SCNT)[5] "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. ... These results indicate that heteroplasmy that originate from donor and recipient mtDNA is maintained in recloned pigs, resulting from SCNT, unlike natural reproduction."

Legislation

This technique essentially results in a clone of the original animal and therefore has been regulated by different countries in many different ways.

Australia

2010 - Cloning Legislative Review Committee Established

An independent committee has been established by the Federal Government to review cloning legislation in Australia (22 December 2010).

The independent Legislation Review Committee for the review of the Prohibition of Human Cloning for Reproduction Act 2002 and the Research Involving Human Embryos Act 2002 was announced today by the Federal Minister for Mental Health and Ageing, Mark Butler.

2006 - Prohibition of Human Cloning for Reproduction and the Regulation of Human Embryo Research Amendment Act 2006 (formerly known as the Patterson Bill) came into effect in June 2007.

2002 - Research Involving Human Embryos Act 2002 and the Prohibition of Human Cloning Act 2002 were passed by Parliament in December 2002.

Links: Cloning Legislative Review Committee Established | NHMRC - Cloning | Australian Statistics

USA

2012 - Human somatic cell nuclear transfer and cloning by The Ethics Committee of the American Society for Reproductive Medicine.[19]

"This document presents arguments that conclude that it is unethical to use somatic cell nuclear transfer (SCNT) for infertility treatment due to concerns about safety; the unknown impact of SCNT on children, families, and society; and the availability of other ethically acceptable means of assisted reproduction. This document replaces the ASRM Ethics Committee report titled, "Human somatic cell nuclear transfer (cloning)," last published in Fertil Steril 2000;74:873-6."

Europe

2008 - Food Safety, Animal Health and Welfare and Environmental Impact of Animals derived from Cloning by Somatic Cell Nucleus Transfer (SCNT) and their Offspring and Products Obtained from those Animals [20] The committee included a number of recommendations in the paper.

"At present there is no indication that clones or their progeny would pose any new or additional environmental risks compared with conventionally bred animals."

References

  1. 1.0 1.1 K D Sinclair, S A Corr, C G Gutierrez, P A Fisher, J-H Lee, A J Rathbone, I Choi, K H S Campbell, D S Gardner Healthy ageing of cloned sheep. Nat Commun: 2016, 7;12359 PubMed 27459299
  2. Jinsong Li, Valentina Greco, Géraldine Guasch, Elaine Fuchs, Peter Mombaerts Mice cloned from skin cells. Proc. Natl. Acad. Sci. U.S.A.: 2007, 104(8);2738-43 PubMed 17299040 | PMC1815251
  3. C Galli, I Lagutina, A Perota, S Colleoni, R Duchi, F Lucchini, G Lazzari Somatic cell nuclear transfer and transgenesis in large animals: current and future insights. Reprod. Domest. Anim.: 2012, 47 Suppl 3;2-11 PubMed 22681293
  4. Carol L Keefer Artificial cloning of domestic animals. Proc. Natl. Acad. Sci. U.S.A.: 2015; PubMed 26195770
  5. 5.0 5.1 5.2 Minhwa Do, Won-Gu Jang, Jeong Hee Hwang, Hoon Jang, Eun-Jung Kim, Eun-Jeong Jeong, Hosup Shim, Sung Soo Hwang, Keon Bong Oh, Sung June Byun, Jin-Hoi Kim, Jeong Woong Lee Inheritance of mitochondrial DNA in serially recloned pigs by somatic cell nuclear transfer (SCNT). Biochem. Biophys. Res. Commun.: 2012, 424(4);765-70 PubMed 22809505
  6. Chao-Bo Li, Zhen-Dong Wang, Zhong Zheng, Li-Li Hu, Shu-Qi Zhong, Lei Lei Number of blastomeres and distribution of microvilli in cloned mouse embryos during compaction. Zygote: 2011, 19(3);271-6 PubMed 20735894
  7. K H Campbell, J McWhir, W A Ritchie, I Wilmut Sheep cloned by nuclear transfer from a cultured cell line. Nature: 1996, 380(6569);64-6 PubMed 8598906
  8. J B Cibelli, S L Stice, P J Golueke, J J Kane, J Jerry, C Blackwell, F A Ponce de León, J M Robl Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science: 1998, 280(5367);1256-8 PubMed 9596577
  9. T Wakayama, A C Perry, M Zuccotti, K R Johnson, R Yanagimachi Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature: 1998, 394(6691);369-74 PubMed 9690471
  10. A Baguisi, E Behboodi, D T Melican, J S Pollock, M M Destrempes, C Cammuso, J L Williams, S D Nims, C A Porter, P Midura, M J Palacios, S L Ayres, R S Denniston, M L Hayes, C A Ziomek, H M Meade, R A Godke, W G Gavin, E W Overström, Y Echelard Production of goats by somatic cell nuclear transfer. Nat. Biotechnol.: 1999, 17(5);456-61 PubMed 10331804
  11. I A Polejaeva, S H Chen, T D Vaught, R L Page, J Mullins, S Ball, Y Dai, J Boone, S Walker, D L Ayares, A Colman, K H Campbell Cloned pigs produced by nuclear transfer from adult somatic cells. Nature: 2000, 407(6800);86-90 PubMed 10993078
  12. Ji Hye Lee, Ju Lan Chun, Keun Jung Kim, Eun Young Kim, Dong-Hee Kim, Bo Myeong Lee, Kil Woo Han, Kang-Sun Park, Kyung-Bon Lee, Min Kyu Kim Effect of Acteoside as a Cell Protector to Produce a Cloned Dog. PLoS ONE: 2016, 11(7);e0159330 PubMed 27428333
  13. Barbara Barboni, Valentina Russo, Sandra Cecconi, Valentina Curini, Alessia Colosimo, Maria Luigia A Garofalo, Giulia Capacchietti, Oriana Di Giacinto, Mauro Mattioli In vitro grown sheep preantral follicles yield oocytes with normal nuclear-epigenetic maturation. PLoS ONE: 2011, 6(11);e27550 PubMed 22132111 | PLoS One.
  14. Eun Young Kim, Min Jee Park, Hyo Young Park, Eun Ji Noh, Eun Hyung Noh, Kyoung Sik Park, Jun Beom Lee, Chang Jin Jeong, Key Zung Riu, Se Pill Park Improved cloning efficiency and developmental potential in bovine somatic cell nuclear transfer with the oosight imaging system. Cell Reprogram: 2012, 14(4);305-11 PubMed 22816525
  15. Jörg P Burgstaller, Pamela Schinogl, Andras Dinnyes, Mathias Müller, Ralf Steinborn Mitochondrial DNA heteroplasmy in ovine fetuses and sheep cloned by somatic cell nuclear transfer. BMC Dev. Biol.: 2007, 7;141 PubMed 18154666 | BMC Dev Biol.
  16. L X Zhao, G P Zhao, R Q Guo, D Zhang, X H Li, H M Zhou DNA methylation status in tissues of sheep clones. Reprod. Domest. Anim.: 2012, 47(3);504-12 PubMed 22039959
  17. Chih-Jie Shen, Winston T K Cheng, Shinn-Chih Wu, Hsiao-Ling Chen, Tung-Chou Tsai, Shang-Hsun Yang, Chuan-Mu Chen Differential differences in methylation status of putative imprinted genes among cloned swine genomes. PLoS ONE: 2012, 7(2);e32812 PubMed 22393450
  18. Yanchang Wei, Jiang Zhu, Yanjun Huan, Zhongfeng Liu, Cairong Yang, Xinmiao Zhang, Yanshuang Mu, Ping Xia, Zhouhua Liu Aberrant expression and methylation status of putatively imprinted genes in placenta of cloned piglets. Cell Reprogram: 2010, 12(2);213-22 PubMed 20677935
  19. Ethics Committee of the American Society for Reproductive Medicine Human somatic cell nuclear transfer and cloning. Fertil. Steril.: 2012, 98(4);804-7 PubMed 22795681
  20. The European Food Safety Authority Journal (2008) 767, 1-49 PDF

Reviews

Carol L Keefer Artificial cloning of domestic animals. Proc. Natl. Acad. Sci. U.S.A.: 2015; PubMed 26195770

Seung-Ick Oh, Chang Kyu Lee, Kyung Jin Cho, Kyung-Ok Lee, Ssang-Goo Cho, Sunghoi Hong Technological progress in generation of induced pluripotent stem cells for clinical applications. ScientificWorldJournal: 2012, 2012;417809 PubMed 22536140

P Chavatte-Palmer, S Camous, H Jammes, N Le Cleac'h, M Guillomot, R S F Lee Review: Placental perturbations induce the developmental abnormalities often observed in bovine somatic cell nuclear transfer. Placenta: 2012, 33 Suppl;S99-S104 PubMed 22000472

Insoo Hyun Moving human SCNT research forward ethically. Cell Stem Cell: 2011, 9(4);295-7 PubMed 21982229

J B Gurdon, Ian Wilmut Nuclear transfer to eggs and oocytes. Cold Spring Harb Perspect Biol: 2011, 3(6); PubMed 21555407



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Cite this page: Hill, M.A. 2018 Embryology Somatic Cell Nuclear Transfer. Retrieved January 21, 2018, from https://embryology.med.unsw.edu.au/embryology/index.php/Somatic_Cell_Nuclear_Transfer

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