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Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon

Nature Methods volume 6, pages 363–369 (2009)Cite this article

Abstract

Induced pluripotent stem cells (iPSCs) have been generated from somatic cells by transgenic expression of Oct4 (Pou5f1), Sox2, Klf4 and Myc. A major difficulty in the application of this technology for regenerative medicine, however, is the delivery of reprogramming factors. Whereas retroviral transduction increases the risk of tumorigenicity, transient expression methods have considerably lower reprogramming efficiencies. Here we describe an efficient piggyBac transposon–based approach to generate integration-free iPSCs. Transposons carrying 2A peptide–linked reprogramming factors induced reprogramming of mouse embryonic fibroblasts with equivalent efficiencies to retroviral transduction. We removed transposons from these primary iPSCs by re-expressing transposase. Transgene-free iPSCs could be identified by negative selection. piggyBac excised without a footprint, leaving the iPSC genome without any genetic alteration. iPSCs fulfilled all criteria of pluripotency, such as pluripotency gene expression, teratoma formation and contribution to chimeras. piggyBac transposon–based reprogramming may be used to generate therapeutically applicable iPSCs.

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Figure 1: Construction of piggyBac transposon–mediated reprogramming vectors.
Figure 2: Generation of primary iPSCs using piggyBac transposons.
Figure 3: Transposon removal from the established iPSC lines.
Figure 4: Characterization of integration-free iPSCs.
Figure 5: Contribution of integration-free iPSCs to somatic tissue and germ line in chimeras.
Figure 6: Schemes to generate transgene-free iPSCs.

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Acknowledgements

We thank R. Banerjee for karyotype analysis, M. Li, C. Kokubu and K. Horie for help, suggestions and comments, and members of Team 82 and the Research Support Facility of the Wellcome Trust Sanger Institute for their support. K.Y. is funded by postdoctoral fellowship of Japan Society for the Promotion of Science. This work is supported by the Wellcome Trust (WT077187).

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Authors and Affiliations

  1. Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK

    Kosuke Yusa, Roland Rad & Allan Bradley

  2. Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, Osaka, Japan

    Junji Takeda

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  1. Kosuke Yusa
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  2. Roland Rad
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Contributions

K.Y. designed and performed experiments, analyzed data and wrote the paper. R.R. and J.T. performed experiments and assisted in writing the paper. A.B. designed experiments, interpreted and assisted in writing the paper.

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Correspondence to Allan Bradley.

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Yusa, K., Rad, R., Takeda, J. et al. Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon. Nat Methods 6, 363–369 (2009). https://doi.org/10.1038/nmeth.1323

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