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

By K. Yusa, R. Rad, J. Takeda, A. Bradley. Nature Methods, 2009

662 citations recorded by the source corpus.

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.

Subfields: Genetics, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/c23589c8d1c185d6d2e156f6536c7bbab2deea2e

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Citations in the corpus

Induced pluripotency: history, mechanisms, and applications. (2010, background): “Transgene-free iPSCs can also be generated with piggyBac transposons, mobile genetic elements that can be introduced into and removed from the host genome by transient expression of transposase (Woltjen et al. 2009; Yusa et al. 2009).”
Senescence impairs successful reprogramming to pluripotent stem cells. (2009, methodology): “In a similar way, two laboratories used piggyBac transposons that can be shuttled in and out of the host genome by transient expression of transposase (Woltjen et al., 2009; Yusa et al., 2009).”
A hyperactive piggyBac transposase for mammalian applications (2011, methodology): “To test if hyPBase could be a useful tool for transposon removal from iPS cells, we transfected two primary iPS cell lines (iPS25 and iPS28) (21) withmPBase and hyPBase and subjected the transfected cells to FIAU selection.”
A Mammalian enhancer trap resource for discovering and manipulating neuronal cell types (2016, methodology): “pPB-UbC.eGFP (Yusa et al., 2009) was used as the backbone for PB plasmids.”
Development of an optimized backbone of FRET biosensors for kinases and GTPases (2011, methodology): “These cDNAs of fluorophores were inserted into the eukaryotic expression vectors pCAGGS (Niwa et al., 1991) and/or pPBbsr, which harbors blasticidin S–resistant gene (Yusa et al., 2009).”