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Somatic Reprograming by Nuclear Transfer

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Epigenetic Reprogramming During Mouse Embryogenesis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2214))

Abstract

Somatic cell nuclear transfer (SCNT) is a powerful technique, although challenging, to study reprograming into the totipotent state of differentiated nuclei in mammals. This procedure was initially applied in farm animals, then rodents, and more recently in primates. Nuclear transfer of embryonic stem cells is known to be more efficient, but many types of somatic cells have now been successfully reprogramed with this procedure. Moreover, SCNT reprograming is more effective on a per cell basis than induced Pluripotent Stem Cells (iPSC) and provides interesting clues regarding the underlying processes. In this chapter, we describe the protocol of nuclear transfer in mouse that combines cell cycle synchronization of the donor cells, enucleation of metaphase II oocyte and Piezo-driven injection of a donor cell nucleus followed by activation of the reconstructed embryos and nonsurgical transfer into pseudo-pregnant mice. Moreover, this protocol includes two facultative steps to erase the epigenetic “memory” of the donor cells and improve chromatin remodeling by histones modifications targeting.

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References

  1. Campbell KHS, McWhir J, Ritchie WA, Wilmut I (1996) Sheep cloned by nuclear transfer from a cultured cell line. Nature 380(6569):64–66. https://doi.org/10.1038/380064a0

    Article  PubMed  CAS  Google Scholar 

  2. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663–676. https://doi.org/10.1016/j.cell.2006.07.024

    Article  CAS  Google Scholar 

  3. Nakagawa M, Koyanagi M, Tanabe K et al (2008) Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol 26:101–106. https://doi.org/10.1038/nbt1374

    Article  PubMed  CAS  Google Scholar 

  4. Kato Y, Tani T, Sotomaru Y et al (1998) Eight calves cloned from somatic cells of a single adult. Science 282:2095–2098. https://doi.org/10.1126/science.282.5396.2095

    Article  PubMed  CAS  Google Scholar 

  5. 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 394:369–374. https://doi.org/10.1038/28615

    Article  PubMed  CAS  Google Scholar 

  6. Chesné P, Adenot PG, Viglietta C et al (2002) Cloned rabbits produced by nuclear transfer from adult somatic cells. Nat Biotechnol 20:366–369. https://doi.org/10.1038/nbt0402-366

    Article  PubMed  Google Scholar 

  7. Zhou Q, Renard JP, Le Friec G et al (2003) Generation of fertile cloned rats by regulating oocyte activation. Science 302:1179. https://doi.org/10.1126/science.1088313

    Article  PubMed  CAS  Google Scholar 

  8. Liu Z, Cai Y, Wang Y et al (2018) Cloning of macaque monkeys by somatic cell nuclear transfer. Cell 172:881–887.e7. https://doi.org/10.1016/j.cell.2018.01.020

    Article  PubMed  CAS  Google Scholar 

  9. Kurosaka S, Nagao Y, Minami N et al (2002) Dependence of DNA synthesis and in vitro development of bovine nuclear transfer embryos on the stage of the cell cycle of donor cells and recipient cytoplasts. Biol Reprod 67:643–647. https://doi.org/10.1095/biolreprod67.2.643

    Article  PubMed  CAS  Google Scholar 

  10. Wells DN, Laible G, Tucker FC et al (2003) Coordination between donor cell type and cell cycle stage improves nuclear cloning efficiency in cattle. Theriogenology 59:45–59. https://doi.org/10.1016/S0093-691X(02)01273-6

    Article  PubMed  CAS  Google Scholar 

  11. Yu Y, Ding C, Wang E et al (2007) Piezo-assisted nuclear transfer affects cloning efficiency and may cause apoptosis. Reproduction 133:947–954. https://doi.org/10.1530/REP-06-0358

    Article  PubMed  CAS  Google Scholar 

  12. Wakayama T, Rodriguez I, Perry ACF et al (2002) Mice cloned from embryonic stem cells. Proc Natl Acad Sci 96:14984–14989. https://doi.org/10.1073/pnas.96.26.14984

    Article  Google Scholar 

  13. Zhou Q, Jouneau A, Brochard V, Adenot P, Renard JP (2001) Developmental potential of mouse embryos reconstructed from metaphase embryonic stem cell nuclei. Biol Reprod 65:412–419. https://doi.org/10.1093/biolreprod/65.2.412

    Article  PubMed  CAS  Google Scholar 

  14. Yamazaki Y, Makino H, Hamaguchi-Hamada K et al (2001) Assessment of the developmental totipotency of neural cells in the cerebral cortex of mouse embryo by nuclear transfer. Proc Natl Acad Sci 98:14022–14026. https://doi.org/10.1073/pnas.231489398

    Article  PubMed  CAS  Google Scholar 

  15. Ogura A, Inoue K, Ogonuki N et al (2005) Production of male cloned mice from fresh, cultured, and cryopreserved immature sertoli cells. Biol Reprod 62:1579–1584. https://doi.org/10.1095/biolreprod62.6.1579

    Article  Google Scholar 

  16. Kishigami S, Mizutani E, Ohta H et al (2006) Significant improvement of mouse cloning technique by treatment with trichostatin A after somatic nuclear transfer. Biochem Biophys Res Commun 340:183–189. https://doi.org/10.1016/j.bbrc.2005.11.164

    Article  PubMed  CAS  Google Scholar 

  17. Miki H, Inoue K, Kohda T et al (2005) Birth of mice produced by germ cell nuclear transfer. Genesis 41:81–86. https://doi.org/10.1002/gene.20100

    Article  PubMed  CAS  Google Scholar 

  18. Kuwayama H, Tanabe Y, Wakayama T, Kishigami S (2017) Birth of cloned mice from vaginal smear cells after somatic cell nuclear transfer. Theriogenology 94:79–85. https://doi.org/10.1016/j.theriogenology.2017.02.012

    Article  PubMed  Google Scholar 

  19. Wakayama S, Ohta H, Hikichi T et al (2008) Production of Healthy Cloned Mice From Bodies Frozen at -20 Degrees C for 16 Years. Proc Natl Acad Sci U S A. Nov 11;105(45):17318–22. https://doi.org/10.1073/pnas.0806166105

  20. Gurdon JB, Wilmut I (2011) Nuclear transfer to eggs and oocytes. Cold Spring Harb Perspect Biol 3:1–14. https://doi.org/10.1101/cshperspect.a002659

    Article  CAS  Google Scholar 

  21. Gall L, Brochard V, Ruffini S et al (2012) Intermediate filaments promote nuclear mechanical constraints during somatic cell nuclear transfer in the mouse. Cell Reprogram 14:497–504. https://doi.org/10.1089/cell.2012.0027

    Article  PubMed  CAS  Google Scholar 

  22. Liu Z, Hai T, Dai X et al (2012) Early patterning of cloned mouse embryos contributes to post-implantation development. Dev Biol 368:304–311. https://doi.org/10.1016/j.ydbio.2012.05.027

    Article  PubMed  CAS  Google Scholar 

  23. Maalouf WE, Liu Z, Brochard V et al (2009) Trichostatin A treatment of cloned mouse embryos improves constitutive heterochromatin remodeling as well as developmental potential to term. BMC Dev Biol 9:1–10. https://doi.org/10.1186/1471-213X-9-11

    Article  CAS  Google Scholar 

  24. Le Bourhis D, Beaujean N, Ruffini S et al (2010) Nuclear remodeling in bovine somatic cell nuclear transfer embryos using MG132-treated recipient oocytes. Cell Reprogram 12:729–738. https://doi.org/10.1089/cell.2010.0035

    Article  PubMed  CAS  Google Scholar 

  25. Jullien J, Vodnala M, Pasque V et al (2017) Gene resistance to transcriptional reprogramming following nuclear transfer is directly mediated by multiple chromatin-repressive pathways. Mol Cell 65:873–884.e8. https://doi.org/10.1016/j.molcel.2017.01.030

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. Ma H, Morey R, O’Neil RC et al (2014) Abnormalities in human pluripotent cells due to reprogramming mechanisms. Nature 511:177–183. https://doi.org/10.1038/nature13551

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Beaujean N, Taylor J, Gardner J et al (2004) Effect of limited DNA methylation reprogramming in the normal sheep embryo on somatic cell nuclear transfer. Biol Reprod 71:185–193. https://doi.org/10.1095/biolreprod.103.026559

    Article  PubMed  CAS  Google Scholar 

  28. Martin C, Beaujean N, Brochard V et al (2006) Genome restructuring in mouse embryos during reprogramming and early development. Dev Biol 292:317–332. https://doi.org/10.1016/j.ydbio.2006.01.009

    Article  PubMed  CAS  Google Scholar 

  29. Pichugin A, Le Bourhis D, Adenot P et al (2010) Dynamics of constitutive heterochromatin: two contrasted kinetics of genome restructuring in early cloned bovine embryos. Reproduction 139:129–137. https://doi.org/10.1530/REP-08-0435

    Article  PubMed  CAS  Google Scholar 

  30. Ribeiro-Mason K, Boulesteix C, Brochard V et al (2012) Nuclear dynamics of histone h3 trimethylated on lysine 9 and/or phosphorylated on serine 10 in mouse cloned embryos as new markers of reprogramming? Cell Reprogram 14:283–294. https://doi.org/10.1089/cell.2011.0071

    Article  PubMed  CAS  Google Scholar 

  31. Chung YG, Matoba S, Liu Y et al (2015) Histone demethylase expression enhances human somatic cell nuclear transfer efficiency and promotes derivation of pluripotent stem cells. Cell Stem Cell 17:758–766. https://doi.org/10.1016/j.stem.2015.10.001

    Article  PubMed  CAS  Google Scholar 

  32. Matoba S, Liu Y, Lu F et al (2014) Embryonic development following somatic cell nuclear transfer impeded by persisting histone methylation. Cell 159:884–895. https://doi.org/10.1016/j.cell.2014.09.055

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Sepulveda-Rincon LP, Del Llano Solanas E, Serrano-Revuelta E et al (2016) Early epigenetic reprogramming in fertilized, cloned, and parthenogenetic embryos. Theriogenology 86:91–98. https://doi.org/10.1016/j.theriogenology.2016.04.022

    Article  PubMed  Google Scholar 

  34. Van Thuan N, Bui HT, Kim JH et al (2009) The histone deacetylase inhibitor scriptaid enhances nascent mRNA production and rescues full-term development in cloned inbred mice. Reproduction 138:309–317. https://doi.org/10.1530/REP-08-0299

    Article  PubMed  CAS  Google Scholar 

  35. Qiu X, Li N, Xiao X et al (2017) Effects of embryo aggregation and PXD101 on the in vitro development of mouse somatic cell nuclear transfer embryos. Cell Reprogram 19:337–343. https://doi.org/10.1089/cell.2017.0027

    Article  PubMed  CAS  Google Scholar 

  36. Boulesteix C, Beaujean N (2015) Fluorescent immunodetection of epigenetic modifications on preimplantation mouse embryos. Methods Mol Biol. 1222:113–26. https://doi.org/10.1007/978-1-4939-1594-1_9.

    Article  CAS  Google Scholar 

  37. Brochard V, Liu Z (2015) Nuclear transfert in mouse. Methods Mol Biol. 1222:1–14. https://doi.org/10.1007/978-1-4939-1594-1_1

  38. Terashita Y, Wakayama S, Yamagata K et al (2012) Latrunculin A can improve the birth rate of cloned mice and simplify the nuclear transfer protocol by gently inhibiting actin polymerization. Biol Reprod 86:1–6. https://doi.org/10.1095/biolreprod.111.098764

    Article  CAS  Google Scholar 

  39. Steele KH, Hester JM, Stone BJ et al (2013) Nonsurgical embryo transfer device compared with surgery for embryo transfer in mice. J Am Assoc Lab Anim Sci 52:17–21

    PubMed  PubMed Central  CAS  Google Scholar 

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Author information

Author notes

  1. Nathalie Beaujean

    Present address: Univ Lyon, Université Lyon 1, Inserm, INRAE, Stem Cell and Brain Research Institute U1208, USC 1361, Bron, France

Authors and Affiliations

  1. Université Paris-Saclay, INRAE, ENVA, BREED U1198, Jouy-en-Josas, France

    Vincent Brochard & Nathalie Beaujean

Authors
  1. Vincent Brochard
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  2. Nathalie Beaujean
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Corresponding author

Correspondence to Nathalie Beaujean .

Editor information

Editors and Affiliations

  1. Institut Curie, CNRS UMR3215/ INSERM U934, Paris Sciences & Lettres Research University (PSL), Paris, France

    Katia Ancelin

  2. Institut Curie, CNRS UMR3215/ INSERM U934, Paris Sciences & Lettres Research University (PSL), Institut de Ge´ne´tique Mole´culaire de Montpellier, Univ Montpellier, CNRS Montpellier, France, Paris, France

    Maud Borensztein

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Brochard, V., Beaujean, N. (2021). Somatic Reprograming by Nuclear Transfer. In: Ancelin, K., Borensztein, M. (eds) Epigenetic Reprogramming During Mouse Embryogenesis. Methods in Molecular Biology, vol 2214. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0958-3_8

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  • DOI: https://doi.org/10.1007/978-1-0716-0958-3_8

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0957-6

  • Online ISBN: 978-1-0716-0958-3

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