期刊
NATURE PROTOCOLS
卷 6, 期 9, 页码 1251-1273出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2011.374
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资金
- Starr Foundation (Tri-Institutional Stem Cell Initiative) [Tri-SCI-018]
- New York State Stem Cell Science, NYSTEM [N08T-060]
- National Heart, Blood and Lung Institute, NHLBI [HL053750]
T he 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.
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