Generation of mouse-induced pluripotent stem cells with plasmid vectors

Nat Protoc. 2010 Mar;5(3):418-28. doi: 10.1038/nprot.2009.231. Epub 2010 Feb 11.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Cell Dedifferentiation / genetics*
  • DNA Primers / genetics
  • Embryonic Stem Cells / cytology
  • Embryonic Stem Cells / metabolism
  • Fibroblasts / cytology
  • Genes, myc
  • Genetic Vectors*
  • Humans
  • Kruppel-Like Transcription Factors / genetics
  • Mice
  • Octamer Transcription Factor-3 / genetics
  • Plasmids / genetics*
  • Pluripotent Stem Cells / cytology*
  • Pluripotent Stem Cells / metabolism
  • SOXB1 Transcription Factors / genetics
  • Transfection

Substances

  • DNA Primers
  • GKLF protein
  • Kruppel-Like Transcription Factors
  • Octamer Transcription Factor-3
  • Pou5f1 protein, mouse
  • SOXB1 Transcription Factors
  • Sox2 protein, mouse