title: Generation of iPS cells using a BacMam multigene expression system.
abstract: Generation of iPS cells from mouse embryonic fibroblasts (MEF) was achieved using a BacMam transduction system containing a polycistronic plasmid expression vector for coincident and optimized expression of four defined reprogramming transcription factors. The sequences for Oct4, Klf4, Sox2 and c-Myc, were cloned as a fusion gene (OKSM) in a single open reading frame (ORF) via self-cleaving 2A peptides and expressed under the control of the CAG promoter. The transduction efficiency of primary MEF cells with BacMam particles carrying CAG-directed Venus reporter gene is 64-98%. After three successive transductions (at intervals of 3 days) of MEF cells with BacMam particles carrying a OKSM or OSKM cassette, the iPS cell colonies are observed in 15-24 days. A single transduction of MEF cells is also effective in generating sufficiently reprogrammed iPS cell lines. The iPS cell lines from colonies picked were positively stained by Nanog, SSEA-1 immunofluorescence and alkaline phosphatase substrate markers. The advantage of using the EOS-S(4+)-EmGFP reporter to identify sufficiently reprogrammed iPS cell lines is discussed by representing experimental results obtained with electroporated plasmids, such as a mixture of 2 tandem OS and KM plasmids and a polycistronic OKSM expression plasmid.
authors: Department of Molecular Biology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.
authors: Yoko
authors: Y
authors: Takata
authors: Hiroe
authors: H
authors: Kishine
authors: Takefumi
authors: T
authors: Sone
authors: Taichi
authors: T
authors: Andoh
authors: Masami
authors: M
authors: Nozaki
authors: Michael
authors: M
authors: Poderycki
authors: Jonathan D
authors: JD
authors: Chesnut
authors: Fumio
authors: F
authors: Imamoto
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D065150:Cellular Reprogramming / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology; D005822:Genetic Vectors / Q000235:genetics*; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism; D051379:Mice; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D016366:Open Reading Frames / Q000235:genetics; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D050814:Octamer Transcription Factor-3; D016271:Proto-Oncogene Proteins c-myc; D055748:SOXB1 Transcription Factors; C527783:Sox2 protein, mouse; D014764:Viral Proteins; C057421:virus protein 2A