Journal: The Journal of Biological Chemistry
Article Title: Proliferation Rate of Somatic Cells Affects Reprogramming Efficiency *
Figure Lengend Snippet: Higher proliferation rate of MEFs correlates with lower reprogramming efficiency. A and B, time course study of the proliferation rate of MEFs and the reprogramming efficiency with OSK and serially diluted c- Myc viruses. MEFs were transduced with OSK plus c-Myc in 5-fold serial dilutions (including zero concentration). The number of total cells ( A ) and the percentage of GFP + cells ( B ) were monitored every other day during the reprogramming process. C, proliferation rates of two OSK-iPS and two OSKM-iPS clones. Cell numbers were normalized to the value obtained on day 1. D, quantitative PCR analysis of the expression levels of Cdk 1 / 2 / 4 in OSK or OSKM iPS clones. The expression level of each gene was normalized to that of β -actin in the same sample and then normalized to MEFs. E, characterization of cell death in OSK- or OSKM-mediated reprogramming using propidium iodide staining and FACS analysis. F and G , MEFs were infected with OSKM or OSK in combination with a Moloney murine leukemia virus 5′ LTR promoter-driven dsRed virus and seeded at different densities in a 96-well plate, the number of GFP + dsRed − colonies were counted at day 14 ( G ), the number of dsRed + cells were recorded at day 6, and the reprogramming efficiency ( H ) was calculated (number of GFP + dsRed − colonies/number of dsRed + cells). *, p
Article Snippet: About 1.5 μg of RNA was used to synthesize cDNA using Random Hexamer Primer and Moloney murine leukemia virus reverse transcriptase (Promega) according to the manufacturer's protocol.
Techniques: Transduction, Concentration Assay, Clone Assay, Real-time Polymerase Chain Reaction, Expressing, Staining, FACS, Infection