Generation of mouse-induced pluripotent stem cells with plasmid vectors.
Reprogramming of somatic cells into pluripotent stem cells has been reported by introducing a combination of several transcription factors (Oct3/4, Sox2, Klf4 and c-Myc). The induced pluripotent stem (iPS) cells from patient's somatic cells could be a useful source for drug discovery and cell transplantation therapies. However, to date, most iPS cells were made using viral vectors, such as retroviruses and lentiviruses. Here we describe an alternative method to generate iPS cells from mouse embryonic fibroblasts (MEFs) by continual transfection of plasmid vectors. This protocol takes around 2 months to complete, from MEF isolation to iPS cell establishment. Although the reprogramming efficiency of this protocol is still low, the established iPS cells are most likely free from plasmid integration. This virus-free technique reduces the safety concern for iPS cell generation and application, and provides a source of cells for the investigation of the mechanisms underlying reprogramming and pluripotency.
Okita
Keisuke
K
Center for iPS Cell Research and Application (CiRA), Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto, Japan. okita@cira.kyoto-u.ac.jp
Hong
Hyenjong
H
Takahashi
Kazutoshi
K
Yamanaka
Shinya
S
D000818:Animals; D001483:Base Sequence; D054337:Cell Dedifferentiation / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology; D016259:Genes, myc; D005822:Genetic Vectors; D006801:Humans; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics; D051379:Mice; D050814:Octamer Transcription Factor-3 / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D055748:SOXB1 Transcription Factors / Q000235:genetics; D014162:Transfection
D017931:DNA Primers; C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D055748:SOXB1 Transcription Factors; C527783:Sox2 protein, mouse