Generating iPS cells from MEFS through forced expression of Sox-2, Oct-4, c-Myc, and Klf4

J Vis Exp. 2008 Apr 7:(14):734. doi: 10.3791/734.

Abstract

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006; Wernig, et al., 2007; Okita, et al., 2007; Maherali, et al., 2007). We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors (Brambrink et al., 2008). Using these inducible constructs, we can derive induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs). In this video, we demonstrate the procedure for the generation of inducible lentiviruses that express the four transcription factors and show how to infect MEFs with these viruses in order to produce iPS cells. By using inducible lentiviruses, the expression of the four factors in controlled by the addition of doxycyline to the culture medium. The advantage of this system over the traditional retroviral infection is the ability to turn the genes on and off so that the kinetics of reprogramming and gene expression requirements can be analyzed in detail.

Publication types

  • Video-Audio Media

MeSH terms

  • Animals
  • DNA-Binding Proteins / biosynthesis
  • DNA-Binding Proteins / genetics*
  • Disease Models, Animal
  • Fibroblasts / metabolism
  • Fibroblasts / pathology
  • Fibroblasts / virology
  • Gene Expression Regulation*
  • Genes, myc
  • Kruppel-Like Factor 4
  • Kruppel-Like Transcription Factors / biosynthesis
  • Kruppel-Like Transcription Factors / genetics*
  • Lentivirus Infections / genetics
  • Lentivirus Infections / pathology
  • Lentivirus Infections / virology
  • Mice
  • Octamer Transcription Factor-3 / biosynthesis
  • Octamer Transcription Factor-3 / genetics*
  • RNA / genetics*
  • SOXB1 Transcription Factors / biosynthesis
  • SOXB1 Transcription Factors / genetics*
  • Salivary alpha-Amylases
  • Stem Cells / cytology
  • Stem Cells / metabolism*
  • Transcription Factors / biosynthesis
  • Transcription Factors / genetics*
  • Zinc Fingers

Substances

  • DNA-Binding Proteins
  • Klf4 protein, mouse
  • Kruppel-Like Factor 4
  • Kruppel-Like Transcription Factors
  • Octamer Transcription Factor-3
  • SOXB1 Transcription Factors
  • Sox2 protein, mouse
  • Transcription Factors
  • RNA
  • Amy1 protein, mouse
  • Salivary alpha-Amylases