title: Simple and efficient method for generation of induced pluripotent stem cells using piggyBac transposition of doxycycline-inducible factors and an EOS reporter system.
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.
authors.affiliation: Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-Cho, Sakyo-Ku, Japan.
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Tsukiyama
authors.forename: Ryota
authors.initials: R
authors.lastname: Asano
authors.forename: Takamasa
authors.initials: T
authors.lastname: Kawaguchi
authors.forename: Narae
authors.initials: N
authors.lastname: Kim
authors.forename: Masayasu
authors.initials: M
authors.lastname: Yamada
authors.forename: Naojiro
authors.initials: N
authors.lastname: Minami
authors.forename: Yasuhide
authors.initials: Y
authors.lastname: Ohinata
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Imai
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D065150:Cellular Reprogramming / Q000235:genetics; D004251:DNA Transposable Elements / Q000235:genetics*; D004318:Doxycycline / Q000494:pharmacology*; D017930:Genes, Reporter / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D000090062:Kruppel-Like Factor 4; D051379:Mice; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D004251:DNA Transposable Elements; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D050814:Octamer Transcription Factor-3; D055748:SOXB1 Transcription Factors; C527783:Sox2 protein, mouse; D004318:Doxycycline