Generation of transgene-free human induced pluripotent stem cells with an excisable single polycistronic vector. Academic Article uri icon

Overview

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.

publication date

  • August 4, 2011

Research

keywords

  • Cell Culture Techniques
  • Induced Pluripotent Stem Cells
  • Lentivirus
  • Transgenes

Identity

Scopus Document Identifier

  • 80052405826

Digital Object Identifier (DOI)

  • 10.1038/nprot.2011.374

PubMed ID

  • 21886095

Additional Document Info

volume

  • 6

issue

  • 9