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.
authors: Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA. papapete@mskcc.org
authors: Eirini P
authors: EP
authors: Papapetrou
authors: Michel
authors: M
authors: Sadelain
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D005822:Genetic Vectors; D015894:Genome, Human; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism; D019076:Transgenes
chemical_list: D017077:Culture Media, Conditioned; C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; C489427:MYC protein, human; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D016271:Proto-Oncogene Proteins c-myc; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors