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  • Video protocol Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4 (Journal of Visualized Experiments )
    Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc. 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.
    http://www.jove.com/index/details.stp?ID=734
    Added: Sat May 03 2008, Hits: 120, Reviews: 0  Write review  
  • Isolation of Nuclei from Somatic Cells PDF file (Philippe Collas Lab, University of Oslo)
    http://collaslab.com/UserFiles/File/Isolation%20of...
    Added: Tue Jan 01 2008, Hits: 123, Reviews: 0  Write review  
  • Reprogramming Somatic Cells in Extract of Undifferentiated Carcinoma Cells PDF file (Philippe Collas Lab, University of Oslo)
    This protocol describes procedures for directing the program of a model human cell line, 293T epithelial kidney cells towards a more pluripotent state. The methods describe (1) the preparation of cells to be reprogrammed (the “donor” cells), (2) the preparation of nuclear and cytoplasmic extracts, (3) the permeabilization of the donor cells, (4) the set up of the reprogramming reaction, and (5) the resealing of the cells.
    http://collaslab.com/UserFiles/File/Nuclear%20repr...
    Added: Tue Jan 01 2008, Hits: 131, Reviews: 0  Write review