Generation of Mouse-Induced Pluripotent Stem Cells by Lentiviral Transduction

Methods Mol Biol. 2019:1940:63-76. doi: 10.1007/978-1-4939-9086-3_5.

Abstract

Terminally differentiated somatic cells can be reprogrammed into an embryonic stem cell-like state by the forced expression of four transcription factors: Oct4, Klf4, Sox2, and c-Myc (OKSM). These so-called induced pluripotent stem (iPS) cells can give rise to any cell type of the body and thus have tremendous potential for many applications in research and regenerative medicine. Herein, we describe (1) a protocol for the generation of iPS cells from mouse embryonic fibroblasts (MEFs) using a doxycycline (Dox)-inducible lentiviral transduction system; (2) the derivation of clonal iPS cell lines; and (3) the characterization of the pluripotent potential of iPS cell lines using alkaline phosphatase staining, flow cytometry, and the teratoma formation assays.

Keywords: Fibroblasts; Lentiviral transduction; Mouse-induced pluripotent stem cells; OKSM; Reprogramming; Teratoma assay.

MeSH terms

  • Animals
  • Cell Differentiation
  • Cells, Cultured
  • Cellular Reprogramming / genetics*
  • Cellular Reprogramming Techniques*
  • Doxycycline / pharmacology
  • Fibroblasts / cytology*
  • Induced Pluripotent Stem Cells / cytology*
  • Induced Pluripotent Stem Cells / virology*
  • Kruppel-Like Factor 4
  • Kruppel-Like Transcription Factors / metabolism
  • Lentivirus / genetics*
  • Mice
  • Octamer Transcription Factor-3 / metabolism
  • Proto-Oncogene Proteins c-myc / metabolism
  • SOXB1 Transcription Factors / metabolism
  • Transduction, Genetic / methods

Substances

  • Klf4 protein, mouse
  • Kruppel-Like Factor 4
  • Kruppel-Like Transcription Factors
  • Myc protein, mouse
  • Octamer Transcription Factor-3
  • Pou5f1 protein, mouse
  • Proto-Oncogene Proteins c-myc
  • SOXB1 Transcription Factors
  • Sox2 protein, mouse
  • Doxycycline