Generating iPS cells from MEFS through forced expression of Sox-2, Oct-4, c-Myc, and Klf4.
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.
Welstead
G Grant
GG
Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, USA. welstead@wi.mit.edu
Brambrink
Tobias
T
Jaenisch
Rudolf
R
D000818:Animals; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D004195:Disease Models, Animal; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology / Q000821:virology; D005786:Gene Expression Regulation; D016259:Genes, myc; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000096:biosynthesis / Q000235:genetics*; D016180:Lentivirus Infections / Q000235:genetics / Q000473:pathology / Q000821:virology; D051379:Mice; D050814:Octamer Transcription Factor-3 / Q000096:biosynthesis / Q000235:genetics*; D012313:RNA / Q000235:genetics*; D055748:SOXB1 Transcription Factors / Q000096:biosynthesis / Q000235:genetics*; D055291:Salivary alpha-Amylases; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics*; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D050814:Octamer Transcription Factor-3; D055748:SOXB1 Transcription Factors; C527783:Sox2 protein, mouse; D014157:Transcription Factors; D012313:RNA; C524799:Amy1 protein, mouse; D055291:Salivary alpha-Amylases