Simple and efficient method for generation of induced pluripotent stem cells using piggyBac transposition of doxycycline-inducible factors and an EOS reporter system

Authors

  • Tomoyuki Tsukiyama,

    1. Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-Cho, Sakyo-Ku, Kyoto 606-8502, Japan
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    • Present address: Laboratory for Pluripotent Stem Cell Studies, RIKEN Center for Developmental Biology (CDB), Kobe, Hyogo 650-0047, Japan.

  • Ryota Asano,

    1. Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-Cho, Sakyo-Ku, Kyoto 606-8502, Japan
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  • Takamasa Kawaguchi,

    1. Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-Cho, Sakyo-Ku, Kyoto 606-8502, Japan
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  • Narae Kim,

    1. Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-Cho, Sakyo-Ku, Kyoto 606-8502, Japan
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  • Masayasu Yamada,

    1. Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-Cho, Sakyo-Ku, Kyoto 606-8502, Japan
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  • Naojiro Minami,

    1. Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-Cho, Sakyo-Ku, Kyoto 606-8502, Japan
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  • Yasuhide Ohinata,

    1. Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, 5 Sanban-Cho, Chiyoda-Ku, Tokyo 102-0075, Japan
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  • Hiroshi Imai

    Corresponding author
    1. Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-Cho, Sakyo-Ku, Kyoto 606-8502, Japan
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  • Communicated by: Hideyuki Okano

imai@kais.kyoto-u.ac.jp

Abstract

PiggyBac (PB) transposition of reprogramming factors (Oct3/4 (O), Sox2 (S), Klf4 (K) and c-Myc) is a safe, nonviral method for generating induced pluripotent stem cells (iPSCs). However, compared with retroviral methods, the reprogramming efficiency of the PB-mediated methods is relatively low. In this study, we describe a simple and efficient system for generating high-quality iPSCs by a single transfection of multiple plasmids that does not require the use of a virus, special instruments or skilled techniques. To improve reprogramming efficiency, we modified the components of the polycistronic 2A vectors used in this study and also investigated the combination of another reprogramming-related factor (L-Myc). By simultaneous transposition of multiple PB vectors containing an EOS (early transposon promoter and Oct3/4 and Sox2 enhancers) reporter and modified polycistronic doxycycline (Dox)-inducible factors, we reprogrammed mouse somatic cells with an efficiency higher than is usually obtained with retroviral methods and we established some iPSC lines that contributed highly to chimeras. By using the Dox-inducible system, we also showed that the appropriate elimination of exogenous-factor expression at appropriate time accelerated the induction of Oct3/4 when a combination of OKS and c-Myc vectors were used.

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