Generation of iPS Cells Using a BacMam Multigene Expression System
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- Takata Yoko
- Department of Molecular Biology, Research Institute for Microbial Diseases, Osaka University
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- Kishine Hiroe
- Department of Molecular Biology, Research Institute for Microbial Diseases, Osaka University
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- Sone Takefumi
- Department of Molecular Biology, Research Institute for Microbial Diseases, Osaka University
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- Andoh Taichi
- Department of Molecular Biology, Research Institute for Microbial Diseases, Osaka University
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- Nozaki Masami
- Department of Cell Biology-Germ Cell Group, Research Institute for Microbial Diseases, Osaka University
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- Poderycki Michael
- Life Technologies Corporation
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- Chesnut Jonathan D.
- Life Technologies Corporation
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- Imamoto Fumio
- Department of Molecular Biology, Research Institute for Microbial Diseases, Osaka University
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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.<br>
Journal
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- Cell Structure and Function
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Cell Structure and Function 36 (2), 209-222, 2011
Japan Society for Cell Biology
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Details 詳細情報について
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- CRID
- 1390001204694265216
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- NII Article ID
- 130004053883
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- NII Book ID
- AA0060007X
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- ISSN
- 13473700
- 03867196
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- NDL BIB ID
- 023442985
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- Text Lang
- en
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- Data Source
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- JaLC
- NDL
- Crossref
- CiNii Articles
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- Abstract License Flag
- Disallowed