Download m49a direct introduction of plasmid into nucleus using

Survey
yes no Was this document useful for you?
   Thank you for your participation!

* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project

Document related concepts
no text concepts found
Transcript
DIRECT INTRODUCTION OF PLASMID INTO NUCLEUS
USING ON-CHIP ELECTROPORATION
Osamu Kurosawa1,2,Yosuke Sumita1, Murat Gel2,3, Hidehiro Oana2,3, Hidetoshi Kotera2,4,
Tomohisa Kato5,Junya Toguchida5 and Masao Washizu1,2,3
1
Dept. Bio Engineering, The University of Tokyo, 2JST CREST,
3
Dept. Mechanical Engineering, The University of Tokyo,
4
Dept. Mechanical Engineering, Kyoto University,
5
Institute for Frontier Medical Sciences, Graduate School of Medicine, Kyoto University,
ABSTRACT
In this paper, we visualized the motion of plasmids within a cell during pulsation using on-chip electroporation and
clarified the direct introduction of plasmids into nucleus by electrophoresis. By making the orifice pitch nearly equal to
nucleus diameter with guaranteeing the field constriction at a micro orifice, higher yield of transfection was experimentally demonstrated.
KEYWORDS: Field constriction, On-chip Electroporation, Plasmids, Cell nucleus
INTRODUCTION
Introduction of foreign genes into cells is a basic process in cellular engineering, including cell reprogramming and
differentiation control through iPS cells. Presently, the retrovirus method is commonly used to create iPS cells, but it is
not suitable for clinical use due to hazardous effects of viral infection. Then, more recently, the method using plasmid instead of retroviral vector has been tried. The problem in using plasmid is inefficiency of its yield.
We have previously reported an on-chip electroporation method based on field constriction at micro-orifices (Fig.1)
[1], which essentially features high-yield and high-survivability due to the fact that the cell membrane voltage can precisely be controlled regardless of cell size, shape or orientation. It has been found that plasmids are transferred into a cell
by electrophoresis[2], and in some cases gene expression was observed within 2 hours after pulsing. Such a quick expression is expected to occur only when the plasmids are directly driven into cell nucleus by electrophoresis. It is expected that direct introduction of plasmid into cell nucleus improves the gene expression yield, because digestion by the
nuclease in the cytoplasm is avoided.
In this paper, we firstly checked the motion of plasmids within a cell during pulsation and next investigated the optimal design of orifice sheet for the on-chip electroporation chip.
Figure 1: Electroporation using field constriction
at a micro-orifice
Figure 2: SEM image of an orifice
EXPRIMENTAL
In order to clarify the motion of plasmids within a cell during pulsation, we made use of a microchip having an orifice
on a vertical wall, whose SEM image is shown in Fig.2. Experimental set up is schematically shown in Fig.3. The device
consists of PDMS chip and microelectrode on a glass coverslip. The PDMS chip was fabricated by single mask photolithography based on self-forming meniscus and PDMS molding [3]. Firstly, the cells were fed into one channel (right-side
channel in Fig.3) and driven toward an orifice by gentle flow. Next, by applying an AC voltage of 1MHz-6Vpeak, the cell
was trapped at the orifice by dielectrophoretic force. Then, plasmid labeled with fluorescent Q-dots (hereafter Qdot-DNA
complex) were fed on the other side of the orifice. Fig.4 shows the bright field image of this instance. Finally, a pulse
voltage (2V-100msec) was applied to the electrodes. Using EB-CCD camera, real-time motion of plasmids during pulsation was observed. To examine the existence part of plasmids introduced into the cell, the depth-wise fluorescence slice
view of the cell was taken.
978-0-9798064-3-8/µTAS 2010/$20©2010 CBMS
217
14th International Conference on
Miniaturized Systems for Chemistry and Life Sciences
3 - 7 October 2010, Groningen, The Netherlands
Figure 4: Bright field image of immobilized cell
Figure 3: Experimental set-up
In order to investigate the optimal design of orifice sheet for the on-chip electroporation chip, we prepared three types
of orifice sheets. The pore size and the pitch are 5-50 microns (No.1), 3-30 microns (No.2), and 2 micron of random
pitch (No.3) respectively, as shown by SEM pictures in Fig.8. Adherent MSC (Mesenchymal Stem Cell) was cultured on
each orifice sheet until nearly-confluent layer was formed, and then GFP plasmid was fed by 4V-200msec pulse. Gene
expression of GFP plasmid was checked with fluorescence microscopy.
RESULTS AND DISCUSSION
In the experiment using the device shown in Fig.3, the cell near the orifice was moved towards the orifice by dielectrophoretic force, by applying AC voltage. A part of the cell membrane invaded into the orifice, and formed the hernia
structure (illustrated in Fig.6). The trapping was made strong enough that the cell tightly sealed the orifice and no leakage
of Qdot-DNA complex occurred. During pulsation, though we could not clarify the motion of each Qdot-DNA complex,
inflow of Qdot-DNA complexes into the cell was observed. Fig.5 (A to D corresponds to depth-wise position in Fig.6)
shows the depth-wise fluorescence and bright field slice view of the cell. Although with a limited resolution, some QdotDNA complexes, which probably failed to pass through the pore, are observed to get stuck near the membrane (nucleus
or cell membrane). Red arrows in the Fig.5 show the position of Qdot-DNA complex. Fig.6 is a schematic re-constructed
from Fig.5, showing that the plasmids are fed into the nucleus, which cannot take place by simple diffusion in the time
scale of the experiment, but most likely to be driven by electrophoresis through nuclear pores.
Figure 5:The depth-wise fluorescence slice image of Qdot-DNA
introduced cell
Figure 6: Schematic view of observed
focal plane
The result that plasmids are transferred directly into nucleus by electroporesis implies that the orifice density should be
chosen in such a way that at least one orifice exists below each nucleus. Fig.7 shows the shape and size of MSC, which
was fluorescently labeled with Calcein. Its length is longer than 100μm,
and width is shorter than 50μm. Probably a diameter of cell nucleus which
spread on the orifice sheet is about 20μm. In Fig.8, schematic drawing of
relative position between typical cell with nucleus and each orifice is
shown. In each orifice sheet, at least one of orifice exists under the cell. But
as for the nucleus, it is not the case. Frequency of the orifice existence under the nucleus increases in order of No.1, No.2, and No.3. Especially an
orifice in the No.3 sheet (Fig.8-c) surely exists right under the nucleus, but
it does not guarantee the field constriction. Therefore, if the yield of GFP
expression depends on the plasmid influx into nucleus, No.2 sheet is expected the highest among them.
Figure 7: Shape and size of MSC
GFP expressions 5 hours after pulsation are shown in Fig.8 a-c. As exlabeled
with green fluorophore
pected, No.2 sheet with an optimized density (Fig.8-b) gives the highest
yield. No.3 sheet also gives good yield (Fig.8-c), but too high orifice densi218
ty reduces field constriction, which made the yield lower. The yield of GFP expression in Fig.8-b is about 50% against
the total cell number on the orifice sheet. It is expected that by optimizing the size and pitch of an orifice, higher yield
transfection is possible.
Figure8: GFP expression on three types of orifice sheets 5 hours after pulsation
CONCLUSION
In conclusion, the electrophoretic effect of plasmid transfection directly into cell nuclei was clarified, and the design
rule for the on-chip electroporation chip was established, through which the yield as high as 50 % was achieved for MSC
cells.
ACKNOWLEDGEMENTS
This research is supported by Scientific Research of Priority Areas, System Cell Engineering by Multi-scale Manipulation, Japanese Ministry of Education and “Development of bio/nano hybrid platform technology towards regenerative
medicine” project of CREST, JST. The photomasks were prepared by the electron-beam facility at the VLSI Design and
Education Centre (VDEC) of the University of Tokyo.
REFERENCES
[1] Osamu Kurosawa et al: "Electroporation through a micro-fabricated orifice and its application to the measurement
of cell response to external stimuli", Measur. Sci. Tech., 17, p.3127-3133, 2006
[2] Osamu Kurosawa et al: "Massively parallel on-chip Electroporation device Designed for long term post-culturing",
µ-TAS 2009, p.570-572, 2009
[3] Murat Gel et al.: "Micro-orifice based cell fusion applied for the creation of cytoplasmic hybrid cells without gene
mixing", µ-TAS 2009, p.654-656, 2009
CONTACT
* M. Washizu, tel: +81-3-5841-6344; [email protected]
219