Generation of transgene-free human induced pluripotent stem cells with an excisable single polycistronic vector Journal Article

  


Authors: Papapetrou, E. P.; Sadelain, M.
Article Title: Generation of transgene-free human induced pluripotent stem cells with an excisable single polycistronic vector
Abstract: The generation of induced pluripotent stem cells (iPSCs) devoid of permanently integrated reprogramming factor genes is essential to reduce differentiation biases and artifactual phenotypes. We describe a protocol for the generation of human iPSCs using a single polycistronic lentiviral vector (pLM-fSV2A) coexpressing OCT4, SOX2, KLF4 and c-MYC; this is flanked by two loxP sites in its long terminal repeats (LTRs). Human iPSC lines are established with an efficiency of up to 1% and screened to select single or low vector copy lines. To deal with potential insertional mutagenesis, the vector integrations are then mapped to the human genome. Finally, the vector is excised by transient expression of Cre recombinase (coexpressed with mCherry) through an integrase-deficient lentiviral vector. Vector-excised iPSC lines maintain all characteristics of pluripotency. This protocol can be used to efficiently derive transgene-free iPSCs from many different starting cell types in approximately 12-14 weeks. © 2011 Nature America, Inc. All rights reserved.
Keywords: controlled study; protein expression; unclassified drug; human cell; nonhuman; protein analysis; animals; mice; cells, cultured; cell differentiation; cell type; genetic vectors; gene mapping; genetic transfection; human genome; myc protein; kruppel-like transcription factors; lentivirus; lentivirus vector; transgene; cell culture techniques; mesenchymal stem cell; pluripotent stem cell; gene dosage; proto-oncogene proteins c-myc; genetic screening; cre recombinase; octamer transcription factor 4; transcription factor sox2; octamer transcription factor-3; soxb1 transcription factors; mutagenesis; transgenes; genome, human; nuclear reprogramming; induced pluripotent stem cells; kruppel like factor 4; long terminal repeat; integrase; culture media, conditioned; single polycistronic lentivirus vector; insertional mutagenesis; loxp site; transient expression
Journal Title: Nature Protocols
Volume: 6
Issue: 9
ISSN: 1754-2189
Publisher: Nature Publishing Group  
Date Published: 2011-01-01
Start Page: 1251
End Page: 1273
Language: English
DOI: 10.1038/nprot.2011.374
PROVIDER: scopus
PUBMED: 21886095
DOI/URL:

http://www.scopus.com/inward/record.url?eid=2-s2.0-80052405826&partnerID=40&md5=3c6734c03c66ffc2e349496dd4f78d44
Notes: --- - "Export Date: 3 October 2011" - "Source: Scopus"
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MSK Authors
  1. Michel W J Sadelain
    583 Sadelain
  2. Eirini P Papapetrou
    24 Papapetrou
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