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Generation of Murine Induced Pluripotent Stem Cells through Transposon-Mediated Reprogramming

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Part of the Methods in Molecular Biology book series (MIMB,volume 2454)

Abstract

The seminal discovery of induced pluripotent stem (iPS) cells through ectopic expression of a cocktail of gene factors (OCT4, SOX2, KLF4, and c-MYC) by the group of Yamanaka was a major breakthrough, gained widespread acclaim and garnered much attention in the field of stem cell science. The iPS cells possess most of the characteristics and advantages of embryonic stem (ES) cells without the association of ethical stigma for their derivation. In addition, these cells can give rise to any cell type of the body and thus have tremendous potential for many downstream applications in research and regenerative medicine. The original method requires viral transduction of several reprogramming factors, which may be associated with an increased risk of oncogenicity and insertional mutagenesis. Nonviral methods for generation of iPS cells through somatic cell reprogramming are powerful tools for establishing in vitro disease models, development of new protocols for treatment of different diseases, and creating transgenic mice models. Here, we present a detailed protocol for the generation of transposon-mediated iPS cells from mouse embryonic fibroblasts (MEFs) and give a short overview of the characterization of the generated iPS cell lines.

Key words

  • Induced pluripotent stem cells
  • Reprogramming
  • Mouse
  • Embryonic fibroblasts
  • Oct4-GFP reporter
  • DNA transposon
  • Sleeping Beauty
  • piggyBac
  • Pluripotency

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  • DOI: 10.1007/7651_2021_350
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References

  1. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663–676

    CAS  CrossRef  Google Scholar 

  2. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T et al (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861–872

    CAS  CrossRef  Google Scholar 

  3. Talluri TR, Kumar D, Glage S, Garrels W, Ivics Z, Debowski K, Behr R, Kues WA (2014) Non-viral reprogramming of fibroblasts into induced pluripotent stem cells by Sleeping Beauty and piggyBac transposons. Biochem Biophys Res Commun 450:581–587

    CAS  CrossRef  Google Scholar 

  4. Kues WA (2016) Transposon-based cellular reprogramming to induced pluripotency. In: eLS. John Wiley & Sons, Ltd., Chichester. https://doi.org/10.1002/9780470015902.a0026889

    CrossRef  Google Scholar 

  5. Robinton DA, Daley GQ (2012) The promise of induced pluripotent stem cells in research and therapy. Nature 481:295–305

    CAS  CrossRef  Google Scholar 

  6. Kumar D, Talluri TR, Anand T, Kues WA (2015a) Induced pluripotent stem cells: mechanisms, achievements and perspectives in farm animals. World J Stem Cells 7(2):315–328

    CrossRef  Google Scholar 

  7. Kumar D, Talluri TR, Anand T, Kues WA (2015b) Transposon-based reprogramming to induced pluripotency. Histol Histopathol 30(12):1397–1409

    CAS  PubMed  Google Scholar 

  8. Kumar D, Anand T, Talluri TR, Kues WA (2020) Potential of transposon-mediated cellular reprogramming towards cell-based therapies. World J Stem Cells 12(7):527–544. https://doi.org/10.4252/wjsc.v12.i7.0000

    CrossRef  PubMed  PubMed Central  Google Scholar 

  9. Gonzalez F, Boue S, Izpisua Belmonte JC (2011) Methods for making induced pluripotent stem cells: reprogramming a la carte. Nat Rev Genet 12:231–242

    CAS  CrossRef  Google Scholar 

  10. Sommer CA, Stadtfeld M, Murphy GJ, Hochedlinger K, Kotton DN, Mostoslavsky G (2009) Induced pluripotent stem cell generation using a single lentiviral stem cell cassette. Stem Cells 27:543–549

    CAS  CrossRef  Google Scholar 

  11. Manno CS et al (2006) Successful transduction of liver in hemophilia by AAV-factor IX and limitations imposed by the host immune response. Nat Med 12:342–347

    CAS  CrossRef  Google Scholar 

  12. Hacein-Bey-Abina S et al (2003) LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 302:415–419

    CAS  CrossRef  Google Scholar 

  13. Hacein-Bey-Abina S et al (2008) Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1. J Clin Invest 118:3132–3142

    CAS  CrossRef  Google Scholar 

  14. Braun CJ et al (2014) Gene therapy for Wiskott-Aldrich syndrome–long-term efficacy and genotoxicity. Sci Transl Med 6:227ra33

    CrossRef  Google Scholar 

  15. Cavazzana-Calvo M et al (2010) Transfusion independence and HMGA2 activation after gene therapy of human beta-thalassaemia. Nature 467:318–322

    CAS  CrossRef  Google Scholar 

  16. Okita K, Ichisaka T, Yamanaka S (2007) Generation of germline-competent induced pluripotent stem cells. Nature 448:313–317

    CAS  CrossRef  Google Scholar 

  17. Grabundzija I, Wang J, Sebe A, Erdei Z, Kajdi R, Devaraj A, Steinemann D, Szuhai K, Stein U, Cantz T, Schambach A, Baum C, Izsvák Z, Sarkadi B, Ivics Z (2013) Sleeping Beauty transposon-based system for cellular reprogramming and targeted gene insertion in induced pluripotent stem cells. Nucleic Acids Res 41:1829–1847

    CAS  CrossRef  Google Scholar 

  18. Anand T, Talluri TR, Kumar D, Garrels W, Mukherjee A, Debowski K, Behr R, Kues WA (2016) Differentiation of induced pluripotent stem cells to lentoid bodies expressing a lens cell-specific fluorescent reporter. PLoS One 11:e0157570

    CrossRef  Google Scholar 

  19. Talluri TR, Kumar D, Glage S, Garrels W, Ivics Z, Debowski K, Behr R, Niemann H, Kues WA (2015) Derivation and characterization of bovine induced pluripotent stem cells by transposon-mediated reprogramming. Cell Reprogram 17:131–140

    CAS  CrossRef  Google Scholar 

  20. Tipanee J, Chai YC, VandenDriessche T, Chuah MK (2017) Preclinical and clinical advances in transposon-based gene therapy. Biosci Rep 37:BSR20160614. https://doi.org/10.1042/BSR20160614. PMID: 29089466

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  21. Ivics Z, Izsvák Z (2010) The expanding universe of transposon technologies for gene and cell engineering. Mob DNA 1:25. https://doi.org/10.1186/1759-8753-1-25. PMID: 21138556

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  22. Narayanavari SA, Chilkunda SS, Ivics Z, Izsvak Z et al (2016) Sleeping beauty transposition: from biology to applications. Crit Rev Biochem Mol Biol 52:18–44

    CrossRef  Google Scholar 

  23. Yoshimizu T, Sugiyama N, De Felice FM, Yeom YI, Ohbo K et al (1999) Germline-specific expression of the Oct-4/green fluorescent protein (GFP) transgene in mice. Develop Growth Differ 41:675–684

    CAS  CrossRef  Google Scholar 

  24. Wood EJ (1983) Molecular cloning. A laboratory manual by T Maniatis, E F Fritsch and J Sambrook. pp 545. Cold Spring Harbor Laboratory, New York. 1982. ISBN 0‐87969‐136‐0. Biochem Educ 11:82

    CrossRef  Google Scholar 

  25. Rols MP, Teissie J (1998) Electropermeabilization of mammalian cells to macromolecules: control by pulse duration. Biophys J 75(3):1415–1423

    CAS  CrossRef  Google Scholar 

  26. De Vry J, Martinez-Martinez P, Losen M, Bode GH, Temel Y, Steckler T, Steinbusch HW, Baets MD, Prickaerts J (2010) Low current-driven microelectroporation allows efficient In Vivo delivery of nonviral DNA into the adult mouse brain. Mol Ther 18(6):1183–1191

    CrossRef  Google Scholar 

  27. Hyder I, Eghbalsaied E, Kues WA (2020) Systematic optimization of square-wave electroporation conditions for bovine primary fibroblasts. BMC Mol Cell Biol 21:9

    CAS  CrossRef  Google Scholar 

  28. Desbaillets I, Ziegler U, Groscurth P, Gassmann M (2000) Embryoid bodies: an in vitro model of mouse embryogenesis. Exp Physiol 85:645–651

    CAS  CrossRef  Google Scholar 

  29. Itskovitz-Eldor J, Schuldiner M, Karsenti D, Eden A, Yanuka O, Amit M et al (2000) Differentiation of human embryonic stem cells into embryoid bodies comprising the three embryonic germ layers. Mol Med 6:88–95

    CAS  CrossRef  Google Scholar 

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Acknowledgments

This work was supported by Netaji Subhas International Fellowship of the Indian Council for Agricultural Research (ICAR) to TR Talluri vide Grant No. F.No. 29-1/2009-EQR/Edn dated 29-09-2011. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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Correspondence to Wilfried A. Kues .

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Talluri, T.R., Kumar, D., Kues, W.A. (2021). Generation of Murine Induced Pluripotent Stem Cells through Transposon-Mediated Reprogramming. In: Nagy, A., Turksen, K. (eds) Induced Pluripotent Stem (iPS) Cells. Methods in Molecular Biology, vol 2454. Humana, New York, NY. https://doi.org/10.1007/7651_2021_350

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  • DOI: https://doi.org/10.1007/7651_2021_350

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2118-9

  • Online ISBN: 978-1-0716-2119-6

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