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

  • Control of inner cells' proportion by asymmetric divisions and ensuing resilience of cloned rabbit embryos[4] "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."
  • 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


More recent papers  
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Search term: Somatic Cell Nuclear Transfer

<pubmed limit=5>Somatic Cell Nuclear Transfer</pubmed>

Older papers  
  • Review - Artificial cloning of domestic animals[5] "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)[6] "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[7] "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."

SCNT Animal Timeline

Cloned dog.jpg

Cloned Dog (2016)[13]

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[14]

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.[15]

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[16]

Several studies have reported that introduction of the somatic nuclei leads to deleterious epigenetic changes including DNA methylation and histone acetylation.[17][18][19]

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[6] has demonstrated a mixed inheritance pattern.

Inheritance of mitochondrial DNA in serially recloned pigs by somatic cell nuclear transfer (SCNT)[6] "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.[20]

"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 [21] 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 Sinclair KD, Corr SA, Gutierrez CG, Fisher PA, Lee JH, Rathbone AJ, Choi I, Campbell KH & Gardner DS. (2016). Healthy ageing of cloned sheep. Nat Commun , 7, 12359. PMID: 27459299 DOI.
  2. Li J, Greco V, Guasch G, Fuchs E & Mombaerts P. (2007). Mice cloned from skin cells. Proc. Natl. Acad. Sci. U.S.A. , 104, 2738-43. PMID: 17299040 DOI.
  3. Galli C, Lagutina I, Perota A, Colleoni S, Duchi R, Lucchini F & Lazzari G. (2012). Somatic cell nuclear transfer and transgenesis in large animals: current and future insights. Reprod. Domest. Anim. , 47 Suppl 3, 2-11. PMID: 22681293 DOI.
  4. Fabrèges D, Daniel N, Duranthon V & Peyriéras N. (2018). Control of inner cells' proportion by asymmetric divisions and ensuing resilience of cloned rabbit embryos. Development , , . PMID: 29567671 DOI.
  5. Keefer CL. (2015). Artificial cloning of domestic animals. Proc. Natl. Acad. Sci. U.S.A. , 112, 8874-8. PMID: 26195770 DOI.
  6. 6.0 6.1 6.2 Do M, Jang WG, Hwang JH, Jang H, Kim EJ, Jeong EJ, Shim H, Hwang SS, Oh KB, Byun SJ, Kim JH & Lee JW. (2012). Inheritance of mitochondrial DNA in serially recloned pigs by somatic cell nuclear transfer (SCNT). Biochem. Biophys. Res. Commun. , 424, 765-70. PMID: 22809505 DOI.
  7. Li CB, Wang ZD, Zheng Z, Hu LL, Zhong SQ & Lei L. (2011). Number of blastomeres and distribution of microvilli in cloned mouse embryos during compaction. Zygote , 19, 271-6. PMID: 20735894 DOI.
  8. Campbell KH, McWhir J, Ritchie WA & Wilmut I. (1996). Sheep cloned by nuclear transfer from a cultured cell line. Nature , 380, 64-6. PMID: 8598906 DOI.
  9. Cibelli JB, Stice SL, Golueke PJ, Kane JJ, Jerry J, Blackwell C, Ponce de León FA & Robl JM. (1998). Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science , 280, 1256-8. PMID: 9596577
  10. Wakayama T, Perry AC, Zuccotti M, Johnson KR & Yanagimachi R. (1998). Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature , 394, 369-74. PMID: 9690471 DOI.
  11. Baguisi A, Behboodi E, Melican DT, Pollock JS, Destrempes MM, Cammuso C, Williams JL, Nims SD, Porter CA, Midura P, Palacios MJ, Ayres SL, Denniston RS, Hayes ML, Ziomek CA, Meade HM, Godke RA, Gavin WG, Overström EW & Echelard Y. (1999). Production of goats by somatic cell nuclear transfer. Nat. Biotechnol. , 17, 456-61. PMID: 10331804 DOI.
  12. Polejaeva IA, Chen SH, Vaught TD, Page RL, Mullins J, Ball S, Dai Y, Boone J, Walker S, Ayares DL, Colman A & Campbell KH. (2000). Cloned pigs produced by nuclear transfer from adult somatic cells. Nature , 407, 86-90. PMID: 10993078 DOI.
  13. 13.0 13.1 Lee JH, Chun JL, Kim KJ, Kim EY, Kim DH, Lee BM, Han KW, Park KS, Lee KB & Kim MK. (2016). Effect of Acteoside as a Cell Protector to Produce a Cloned Dog. PLoS ONE , 11, e0159330. PMID: 27428333 DOI.
  14. Barboni B, Russo V, Cecconi S, Curini V, Colosimo A, Garofalo ML, Capacchietti G, Di Giacinto O & Mattioli M. (2011). In vitro grown sheep preantral follicles yield oocytes with normal nuclear-epigenetic maturation. PLoS ONE , 6, e27550. PMID: 22132111 DOI.
  15. Kim EY, Park MJ, Park HY, Noh EJ, Noh EH, Park KS, Lee JB, Jeong CJ, Riu KZ & Park SP. (2012). Improved cloning efficiency and developmental potential in bovine somatic cell nuclear transfer with the oosight imaging system. Cell Reprogram , 14, 305-11. PMID: 22816525 DOI.
  16. Burgstaller JP, Schinogl P, Dinnyes A, Müller M & Steinborn R. (2007). Mitochondrial DNA heteroplasmy in ovine fetuses and sheep cloned by somatic cell nuclear transfer. BMC Dev. Biol. , 7, 141. PMID: 18154666 DOI.
  17. Zhao LX, Zhao GP, Guo RQ, Zhang D, Li XH & Zhou HM. (2012). DNA methylation status in tissues of sheep clones. Reprod. Domest. Anim. , 47, 504-12. PMID: 22039959 DOI.
  18. Shen CJ, Cheng WT, Wu SC, Chen HL, Tsai TC, Yang SH & Chen CM. (2012). Differential differences in methylation status of putative imprinted genes among cloned swine genomes. PLoS ONE , 7, e32812. PMID: 22393450 DOI.
  19. Wei Y, Zhu J, Huan Y, Liu Z, Yang C, Zhang X, Mu Y, Xia P & Liu Z. (2010). Aberrant expression and methylation status of putatively imprinted genes in placenta of cloned piglets. Cell Reprogram , 12, 213-22. PMID: 20677935 DOI.
  20. Ethics Committee of the American Society for Reproductive Medicine. (2012). Human somatic cell nuclear transfer and cloning. Fertil. Steril. , 98, 804-7. PMID: 22795681 DOI.
  21. The European Food Safety Authority Journal (2008) 767, 1-49 PDF

Reviews

Keefer CL. (2015). Artificial cloning of domestic animals. Proc. Natl. Acad. Sci. U.S.A. , 112, 8874-8. PMID: 26195770 DOI.

Oh SI, Lee CK, Cho KJ, Lee KO, Cho SG & Hong S. (2012). Technological progress in generation of induced pluripotent stem cells for clinical applications. ScientificWorldJournal , 2012, 417809. PMID: 22536140 DOI.

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

Hyun I. (2011). Moving human SCNT research forward ethically. Cell Stem Cell , 9, 295-7. PMID: 21982229 DOI.

Gurdon JB & Wilmut I. (2011). Nuclear transfer to eggs and oocytes. Cold Spring Harb Perspect Biol , 3, . PMID: 21555407 DOI.

Articles

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August 2012 "somatic cell nuclear transfer" All (1883) Review (360) Free Full Text (585) Published in 2012 (125)

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Cite this page: Hill, M.A. (2018, December 17) Embryology Somatic Cell Nuclear Transfer. Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Somatic_Cell_Nuclear_Transfer

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© Dr Mark Hill 2018, UNSW Embryology ISBN: 978 0 7334 2609 4 - UNSW CRICOS Provider Code No. 00098G