Generation of iPS Cells Using a BacMam Multigene Expression System

• Takata Yoko

  Department of Molecular Biology, Research Institute for Microbial Diseases, Osaka University

• Kishine Hiroe

  Department of Molecular Biology, Research Institute for Microbial Diseases, Osaka University

• Sone Takefumi

  Department of Molecular Biology, Research Institute for Microbial Diseases, Osaka University

• Andoh Taichi

  Department of Molecular Biology, Research Institute for Microbial Diseases, Osaka University

• Nozaki Masami

  Department of Cell Biology-Germ Cell Group, Research Institute for Microbial Diseases, Osaka University

• Poderycki Michael

  Life Technologies Corporation

• Chesnut Jonathan D.

  Life Technologies Corporation

• Imamoto Fumio

  Department of Molecular Biology, Research Institute for Microbial Diseases, Osaka University

抄録

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>

収録刊行物

• Cell Structure and Function

  Cell Structure and Function 36 (2), 209-222, 2011

  日本細胞生物学会

キーワード

• baculovirus vector
• BacMam
• Multisite Gateway
• induced pluripotent stem cell
• polycistronic vector

詳細情報

• CRID: 1390001204694265216
• NII論文ID: 130004053883
• NII書誌ID: AA0060007X
• DOI: 10.1247/csf.11008
• ISSN: 13473700
• ISSN: 03867196
• NDL書誌ID: 023442985
• Web Site: https://ndlsearch.ndl.go.jp/books/R000000004-I023442985
• Web Site: http://www.jstage.jst.go.jp/article/csf/36/2/36_11008/_pdf
• Web Site: https://search.jamas.or.jp/link/ui/2013092025
• 本文言語コード: en
• データソース種別:
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  • NDL
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