Signal Transduction I Receptors & Ligands. Signal Transduction.
Protein Transduction into Eukaryotic Cells using Non … detail of the principle is not clarified....
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I. INTRODUCTION Induced pluripotent stem (iPS) cells have attracted attention recently in the field of regenerative medicine. It is known that there are mainly two types of the way to create iPS cells. One way is “Gene transduction method”, and the other one is “Protein transduction method” [1, 2]. The former is made by introducing genes into cells using retroviral virus vectors. However, all of the methods developed to date still involve the use of genetic materials and thus the potential for unexpected genetic modifications or malignant alterations by the exogenous sequences in the target cells (see Fig. 1) [3]. On the other hand, the latter is made by introducing appropriate proteins functioning in remodeling chromatin structure directly, into cells directly [4]. Therefore, it can avoid unexpected genetic modifications or malignant alterations (see Fig. 2). However, there is a problem that transduction of proteins is often inefficient. In addition, the detail of the principle is not clarified. Atmospheric pressure plasma jet has been attracted in the field of medical application, especially for disinfection, sterilization, tooth whitening and so on [5- 13]. The plasma can be touched by a bare hand without any feeling of electrical shock or warmth (see Fig. 3) [14]. Furthermore, the plasma is a non-thermal, high pressure, uniform glow plasma discharge that produces a high velocity effluent stream of highly reactive chemical species. While passing through the plasma, the feed gas becomes excited, dissociated or ionized by electron impact. Once the gas exits the discharge volume, ions and electrons are rapidly lost by recombination, but the fast-flowing effluent still contains neutral metastable species (e.g., O * 2 , He * ) and radicals (e.g., O, OH) [15]. In this paper, a simple atmospheric pressure plasma jet device, which was recently developed in our Protein Transduction into Eukaryotic Cells using Non-thermal Plasma N. Takamura 1 , D. Wang 2 , D. Seki 1 , T. Namihira 3 , K. Yano 3 , H. Saitoh 1 , and H. Akiyama 1 1 Graduate School of Science and Technology, Kumamoto University, Japan 2 Priority Organization for Innovation and Excellence, Kumamoto University, Japan 3 Bioelectrics 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 Corresponding author: Douyan Wang e-mail address: [email protected] Presented at the 2nd International Symposium on New Plasma and Electrical Discharge Applications and on Dielectric Materials (ISNPEDADM), in November 2011 Retrovirus High!! Cancer Risk Virus infection Gene expression Vector(DNA ) iPS cells Virus gene insertion to chromosome Retrovirus High!! Cancer Risk Virus infection Gene expression Vector(DNA ) iPS cells Virus gene insertion to chromosome Fig. 1. Idea of “Gene transduction method”. Protein Low!! Cancer Risk Protein infusion iPS cells Protein insertion to the target cells Protein Low!! Cancer Risk Protein infusion iPS cells Protein insertion to the target cells Fig. 2. Idea of “Protein transduction method”. Takamura et al. 59
Transcript of Protein Transduction into Eukaryotic Cells using Non … detail of the principle is not clarified....
I. INTRODUCTION
Induced pluripotent stem (iPS) cells have attracted attention recently in the field of regenerative medicine. It is known that there are mainly two types of the way to create iPS cells. One way is “Gene transduction method”, and the other one is “Protein transduction method” [1, 2]. The former is made by introducing genes into cells using retroviral virus vectors. However, all of the methods developed to date still involve the use of genetic materials and thus the potential for unexpected genetic modifications or malignant alterations by the exogenous sequences in the target cells (see Fig. 1) [3]. On the other hand, the latter is made by introducing appropriate proteins functioning in remodeling chromatin structure directly, into cells directly [4]. Therefore, it can avoid unexpected genetic modifications or malignant alterations (see Fig. 2). However, there is a problem that transduction of proteins is often inefficient. In addition, the detail of the principle is not clarified. Atmospheric pressure plasma jet has been attracted in the field of medical application, especially for disinfection, sterilization, tooth whitening and so on [5-13]. The plasma can be touched by a bare hand without any feeling of electrical shock or warmth (see Fig. 3) [14]. Furthermore, the plasma is a non-thermal, high pressure, uniform glow plasma discharge that produces a high velocity effluent stream of highly reactive chemical
species. While passing through the plasma, the feed gas becomes excited, dissociated or ionized by electron impact. Once the gas exits the discharge volume, ions and electrons are rapidly lost by recombination, but the fast-flowing effluent still contains neutral metastable species (e.g., O*2 , He*) and radicals (e.g., O, OH) [15]. In this paper, a simple atmospheric pressure plasma jet device, which was recently developed in our
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
Corresponding author: Douyan Wang e-mail address: [email protected] Presented at the 2nd International Symposium on New Plasma and Electrical Discharge Applications and on Dielectric Materials (ISNPEDADM), in November 2011
Retrovirus
High!!
Cancer Risk
Virusinfection
GeneexpressionVector(DNA) iPS cells
Virus gene insertion to chromosome
Retrovirus
High!!
Cancer Risk
Virusinfection
GeneexpressionVector(DNA) iPS cells
Virus gene insertion to chromosome
Fig. 1. Idea of “Gene transduction method”.
Protein
Low!!
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Proteininfusion
iPS cells
Proteininsertion to
the target cells
Protein
Low!!
Cancer Risk
Proteininfusion
iPS cells
Proteininsertion to
the target cells
Fig. 2. Idea of “Protein transduction method”.
Takamura et al. 59
laboratory, is used to this study in order to improve the transduction efficiency of general “Protein transduction method”.
II. METHODOLOGY
The atmospheric pressure plasma jet system and its
schematic diagram for protein transduction study are shown in Figs. 4 and 5, respectively. Furthermore, Table I shows the experimental conditions of the atmospheric pressure plasma jet system. The atmospheric pressure plasma jet system consists of a low frequency high voltage power supply, a glass tube, a high voltage electrode and a ground electrode placed on the tube with a certain distance separation. The applied voltage and the operating frequency were approximately 10 kV and 10 kHz, respectively. The atmospheric pressure plasma jet appears under the outlet of the glass tube has length of 55 mm maximum.
In this study, HeLa cells were used for protein transduction. The target protein which aimed to be induced to the HeLa cells was the green fluorescent proteins (GFP-7R) fused to the protein transduction domains (PTD), which were prepared in our laboratory.
In this study, irradiating time duration of the plasma jet were varied from 0 to 120 seconds. The distance between the high voltage electrode and the bottom of dish were fixed at 40 mm. GFP-7R fused to PTD were added to the culture medium in advance.
III. RESULTS Fig. 6 shows the fluorescent images of HeLa celles
observed by inverted microscope (ECLIPSE TE2000-S, Nikon, Japan) after different irradiation time duration by atmospheric pressure plasma jet. Upper blue color images show the results of DAPI stained, which indicate
the position of nucleus. Lower green color images indicate the result of GFP-7R transduction into the HeLa cells. From Fig. 6 (a) and (b), it is clear that no green fluorescent can be observed, which means GFP-7R was not introduced into HeLa cells. In case of Fig. 6 (c), green fluorescent was successfully obtained within the nucleus by irradiating atmospheric pressure plasma jet for 30 seconds. On the other hand, strong green fluorescent was observed in Fig. 6 (d). This can be
He
N2+
N2*He*
O2-
UV
electron
He
N2+
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Fig. 3. Atmospheric pressure plasma jet used in this study.
Fig. 4. Image of the atmospheric pressure plasma jet system.
Glass tube
Low frequencyHigh voltage supply
HeLa cells
Culture medium
electrode
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Glass tube
Low frequencyHigh voltage supply
HeLa cellsHeLa cells
Culture medium
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Fig. 5. Schematic diagram of experimental setup for protein
transduction.
TABLE I EXPERIMENTAL CONDITIONS OF ATMOSPHERIC PRESSURE
PLASMA JET SYSTEM USED IN THIS STUDY
Low frequency high voltage power supply
Operating Frequency: 10 kHz Output Voltage: 10 kV
Inside diameter of glass tube 3 mm
Distance between electrodes 30 mm
Helium gas conditions Flow rate: 3.0 L/min Pressure: 0.1 MPa
Length of the plasma jet 55 mm Max.
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presumed that dead cells and its membrane involved the GFP-7R then resulted the bright green color.
Table II shows the experimental results of GFP-7R transduction into HeLa cells by irradiating atmospheric pressure plasma jet. It finds that 30 seconds is the efficient time of plasma irradiation for GFP-7R transduction into HeLa cells without causing cell death.
IV. DISCUSSION
In this study, it is deduced that atmospheric pressure plasma jet can be a useful tool to introduce protein into eukaryotic cells. The irradiation time of plasma is a critical parameter to keep the cells alive and result a successful protein introduction effect. To achieve higher transduction efficiency, it is necessary to investigate the amount and composition of the culture medium, distance between electrode and the dish, parameters of plasma jet and so on. At the same time, it is an important issue to study the mechanism of plasma assist protein introduction. At this point, analysis of culture medium after plasma irradiation, study of radical formations in plasma and medium, composition of PTD, etc. are required. Results of this study gave us a new idea of using atmospheric pressure plasma jet for medical purpose, also remaining subjects to understand the details
of interaction between plasma and cell responses. Further studies are required to extend this novel research topic.
V. CONCLUSION
Atmospheric pressure plasma jet was used to assist
the protein transduction into eukaryotic cells. Experimental results showed that GFP-7R was successfully introduced into HeLa cells under a certain plasma irradiation time. Higher efficiency improvement, interaction between culture medium, plasma and cells, mechanism of protein transduction using plasma are required to explain the details of this study.
ACKNOWLEDGMENT
This research was supported by the Kumamoto University Global COE (Center of Excellence) Program, Global Initiative Center for Pulsed Power Engineering.
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(a) 0 second (b) 10 second (c) 30 second (d) 60second
Fig. 6. Typical fluorescent images of HeLa cells observed by inverted microscope after different irradiation time duration by atmospheric
pressure plasma jet. Upper blue color images show the results of DAPI stained, which indicate the position of nucleus. Lower green color images indicate the result of GFP-7R transduction into the HeLa cells.
TABLE II EXPERIMENTAL RESULTS OF GFP-7R TRANSDUCTION INTO HELA CELLS BY IRRADIATING ATMOSPHERIC PRESSURE PLASMA JET
Irradiation time of plasma jet GFP-7R transduction into HeLa cells
0 sec Not introduced
10 sec Not introduced
20 sec Not introduced
30 sec Introduced
60 sec Cell death
80 sec Cell death
120 sec Cell death
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