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.
Video Link
The video component of this article can be found at https://www.jove.com/video/734/
Protocol
Step 1:
Thaw out 293 cells and plate for lentivirus production.
1. Prepare 9 ml of DMEM+ medium in a 15-ml tube. 2. Remove a vial of frozen 293 stocks from the liquid nitrogen tank and put the vial in a 37°C water bath until most (but not all) cells are thawed. 3. Remove cells from freezing vial and place into the 15ml tube from step 1. 4. Centrifuge at 180g for 5 min, and then discard the supernatant. 5. Resuspend the cells with 10 ml of DMEM+ medium, and transfer to a gelatin-coated 100-mm dish. Incubate the cells in a 37°C, 5% CO 2 incubator until they are 80-90% confluent. 6. Passage of the 293 cells: wash cells with PBS, aspirate the PBS, and add 4 ml per 10-cm dish of 0.05% trypsin/0.53 mM EDTA, and incubate for 1 min at 37°C. 7. Detach cells from dishes by tapping, resuspend with 10 ml DMEM+ medium, and transfer to a 15-ml tube. Centrifuge at 180g for 5 min, and aspirate the supernatant. 8. Add appropriate volume of DMEM+ medium, and break up the cells into a single cell suspension by pipetting up and down several times. 9. Count the number of cells and adjust the concentration to 8x10 5 cells per ml with FP medium.
10. Seed cells at 8x10 6 cells (10 ml) per 100-mm culture dish, and incubate overnight at 37°C, 5% CO 2 .
Step 2:
Transfect 293 cells with lentivirus plasmids and harvest virus 1. For each 10-cm plate, use 10 µg of FUW-tetO-cDNA (Oct4, Sox2, Klf4 or c-Myc) lentivirus backbone with 2.5ug of pMD-G and 7.5 µg of pPax2. 2. While aliquoting the three plasmids into a tube, deliver 30µl of Fugene 6 transfection reagent into a second tube containing 500µl of DMEM without serum and mix gently by finger tapping and incubate for 5 min at room temperature. 3. Add the DNA mix drop-by-drop into the Fugene 6/DMEM-containing tube, mix gently by finger tapping and incubate for 20 min. 4. Add the DNA/Fugene 6 complex dropwise into the dish of 293's, and incubate overnight at 37°C, 5% CO 2 . 5. Next morning: Aspirate the transfection reagent-containing medium, add 5 ml of fresh ES cell medium, and return the cells to the incubator. 6. At 48 hours and 72 hours after transfection, collect the medium from the 293's by using a 10-ml sterile disposable syringe, filtering it through a 0.45-µm pore size cellulose acetate filter, and transferring into a 15-ml tube. 7. Concentrate the virus supernatant by ultracentrifugation. Resuspend the virus pellet in desired volume and make amixture of equal parts of the medium containing Oct-3/4-, Sox2-, Klf4-and c-Myc-lentiviruses.

{'paper_id': '965799', 'title': 'Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4', 's2_url': 'https://api.semanticscholar.org/CorpusID:965799', 'source': 'S2ORC'}