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 expresse...

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Bibliographic Details
Published in:Journal of visualized experiments 2008-04 (14)
Main Authors: Welstead, G Grant, Brambrink, Tobias, Jaenisch, Rudolf
Format: Article
Language:English
Subjects:
Animals
Cell Biology
Disease Models, Animal
DNA-Binding Proteins - biosynthesis
DNA-Binding Proteins - genetics
Fibroblasts - metabolism
Fibroblasts - pathology
Fibroblasts - virology
Gene Expression Regulation
Genes, myc
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
Salivary alpha-Amylases
SOXB1 Transcription Factors - biosynthesis
SOXB1 Transcription Factors - genetics
Stem Cells - cytology
Stem Cells - metabolism
Transcription Factors - biosynthesis
Transcription Factors - genetics
Zinc Fingers
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Summary: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.
ISSN:1940-087X
1940-087X
DOI:10.3791/734