Induced pluripotent stem (iPS) cells have been generated from mouse and human somatic cells by etopic expression of transcription factors. iPS cells are indistinguishable from ES cells in terms of morphology and stem cell marker expression. Moreover, mouse iPS cells give rise to chimeric mice that are competent for germline transmission. However, mice derived from iPS cells often develop tumors. Furthermore, the low efficiency of iPS cell generation is a big disadvantage for mechanistic studies. Nonviral plasmid‐based vectors are free of many of the drawbacks that constrain viral vectors. The histone deacetylase inhibitor valproic acid (VPA) has been shown to improve the efficiency of mouse and human iPS cell generation, and vitamin C (Vc) accelerates gene expression changes and establishment of the fully reprogrammed state. The MEK inhibitor PD0325901 (Stemgent) has been shown to increase the efficiency of the reprogramming of human primary fibroblasts into iPS cells. In this report, we described the generation of mouse iPS cells devoid of exogenous DNA by the simple transient transfection of a nonviral vector carrying 2A‐peptide‐linked reprogramming factors. We used VPA, Vc, and the MEK inhibitor PD0325901 to increase the reprogramming efficiency. The reprogrammed somatic cells expressed pluripotency markers and formed EBs.

ABSTRACT   INTRODUCTION   EXPERIMENTAL PROCEDURES    Mouse Embryonic Fibroblast Isolation and iPS Cell Culture    Plasmid Preparation and Infection    AP Staining    Immunocytochemistry    Quantitative Real-Time Polymerase Chain Reaction (Real-Time PCR) Analysis    Embryoid Body Formation Analysis    Statistical Analyses   RESULTS    Induced Pluripotent Stem Cell Generation using a Nonviral Vector    Virus-Free Induced Pluripotent Stem Cells Express Pluripotency Marker Gene    Characterization of iPS Cells Generated using Nonviral Vectors    In Vitro Differentiation   DISCUSSION   REFERENCES

• Si Jun Park(School of Life Science and Biotechnology, College of Natural Sciences, Kyungpook National University)
• Mi Jung Shin(School of Life Science and Biotechnology, College of Natural Sciences, Kyungpook National University)
• Byoung Boo Seo(Dept. of Animal Resources, College of Life & Environmental Science, Daegu University)
• Humdai Park(Dept. of Biotechnology, College of Engineering, Daegu University)
• Du Hak Yoon(School of Life Science and Biotechnology, College of Natural Sciences, Kyungpook National University)
• Zae Young Ryoo(School of Life Science and Biotechnology, College of Natural Sciences, Kyungpook National University) Corresponding author