Protein Transduction into Eukaryotic Cells using Non-thermal Plasma
N. Takamura1, D. Wang2, D. Seki1, T. Namihira3, K. Yano3, H. Saitoh1, and H. Akiyama1
1Graduate School of Science and Technology, Kumamoto University, Japan
2Priority Organization for Innovation and Excellence, Kumamoto University, Japan
3Bioelectrics Research Center, Kumamoto University, Japan
Abstract— Groundbreaking work demonstrated that ectopic expression of four transcription factors, Oct4, Klf4, Sox2, and c-Myc, referred to as Yamanaka factors, could reprogram mammalian somatic cells to induced pluripotent stem (iPS) cells. To address the safety issues arose from harboring integrated exogenous sequences in the target cell genome, a number of modified genetic methods have been developed and produced iPS cells with potentially reduced risks. However, all of the methods developed to date still involve the use of genetic materials and the potential for unexpected genetic modifications by the exogenous sequences in the target cells. One possible way to avoid introducing exogenous genetic modifications to target cells would be to deliver appropriate proteins functioning in remodeling chromatin structure directly into cells, rather than relying on the transcription from delivered genes. However, using current strategies, induction of proteins is often inefficient. In this study, we show the experimental result that GFP-7R proteins (green fluorescent protein fused to poly-arginine) were promoted delivering to the human cultured cells (HeLa cells) by radiating atmospheric-pressure plasma jet which is used to apply it to biotechnology. GFP-7R is a polypeptide sequence previously documented to stimulate incorporation into cells. The atmospheric pressure plasma jet device consists of a low frequency high voltage power supply, a glass tube, a high voltage electrode and ground electrode placed on the tube with a certain distance separation. The applied voltage and the operating frequency are approximately 10 kV and 10 kHz, respectively. Plasma jet appears under the outlet of the tube has length of 55 mm maximum. The plasma treated cells showed significantly increased green fluorescent signals in comparison with no plasma treated cells, suggesting that GFP-7R proteins were delivered more efficiently to plasma treated cells than non-treated cells.
Keywords— Atmospheric-pressure plasma jet, HeLa cells, iPS cells, protein transduction