JP2002142763A - Method and device for transferring chemical substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method and a device therefor capable of continuously treating a large quantity of cells selectively and highly efficiently through targeting individual cells and intended for transferring a desired substance such as a gene into specific cells. SOLUTION: This method and the device therefor for transferring a substance into cells comprise irradiating a system having the cells and the substance such as a gene to be transferred thereinto with laser beams of absorption wavelength for the cells; wherein, more particularly, the system consists of a mobile phase and moves continuously to a laser beam irradiation point to effect transferring the substance into the cells continuously.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for introducing a substance such as a gene, a protein, or a drug into cells, and an apparatus therefor. More specifically, the present invention relates to a method and an apparatus for introducing a gene into cells using elastic expansion caused by thermal stress excited by a free electron laser (FEL) having a wavelength that resonates with infrared absorption of a cell membrane or the like. In addition, the present invention provides a method for continuously moving a system comprising cells and a substance to be introduced into the cells in a certain direction and irradiating a predetermined position of the mobile phase with a free electron laser (FEL). The present invention relates to a method and an apparatus for introducing a substance into a material.

[0002]

2. Description of the Related Art In recent years, in the fields of medicine, pharmacy, genetic engineering, and molecular biology, a gene having a macromolecule such as a protein has been introduced into cells of animals and plants for drug evaluation, gene recombination, gene therapy, and development of new species. There is a lot going on. As a method for gene transfer, a microinjection method in which a glass pipe is microscopically manipulated and the tip of the glass pipe is inserted directly into a cell to insert the gene, or electroporation, which applies an electric shock to the cell to deform the cell and introduce the gene Methods, particle gun methods, biological methods using viruses such as adenovirus and lambda phage are known.

[0003] However, in a biological method using a virus or the like, a gene can be introduced only into a specific cell such as a mature lymphocyte, and there is a concern about virus infection. In a method using physical means such as a microinjection method and a particle gun method, cells can be individually treated, but the selectivity of the cells is low and the gene cannot be efficiently incorporated. In addition, in the electroporation method, the enzymatic method, etc., there is a probability that a gene can be introduced at a rate of one or less in thousands to tens of thousands of cells, and the transfection efficiency is extremely low. There is a problem that can not be.

In recent years, methods for physically introducing genes and the like into cells using a laser have been developed. For example, a method has been developed in which a cell in which an antibody having a light absorber bound to a cell surface antigen is bound to the cell and irradiated with laser light from a free electron laser to introduce the gene into the cell (Japanese Patent Laid-Open No. 11-1999). No. 46761). However, in this method, an antibody to which a light absorber is bound must be prepared and bound to the cell surface, which requires not only a complicated operation but also the antibody bound to the cell surface after gene transfer. It was not a practical method. Also, a method using a shock wave has been developed, for example,
A method for containing a liquid that generates a shock wave by a pulse laser in an ultraviolet region or the like, and having a chamber means or a balloon means for transmitting the generated shock wave to the outside, and introducing the gene into cells by this shock (Japanese Patent Laid-Open No. 11-28086) Have been reported. However, this method has no cell selectivity, and the size of a device such as a shock wave generator and a transmission device is increased, so that a gene cannot be efficiently introduced into a specific cell.

[0005] As described above, various methods have been developed as a method for introducing a gene into cells, but none of these methods has necessarily provided sufficient selectivity to cells and gene introduction efficiency. In addition, this method is not a method that can continuously treat a large amount of individual cells in a short time.

[0006] On the other hand, a new mechanism that allows a desired substance to penetrate a target biological membrane in a minimally invasive manner by utilizing an impact reaction excited by a free electron laser (FEL) having a wavelength that resonates with infrared absorption of a cell membrane or the like. Has been proposed.
For example, a method of irradiating an artery with arteriosclerosis with a free electron laser (FEL) to selectively remove cholesterol ester from a lesion (Awazu, K., et al., (199)
7) IONICS 23 (11), 89-94), FITC-
A method of introducing dextran (Szoka, F., et al., (198
0) Biochim. Biophys. Acta 601, 559-571). This method utilizes the elastic expansion caused by thermal stress when a living body is irradiated with a free electron laser in the infrared region, and it is possible to physically introduce a substance into a living body or cell in a minimally invasive manner. It is thought to come out.

[0007]

SUMMARY OF THE INVENTION In order to solve the above-mentioned problems when introducing a gene into cells, the present inventors have proposed a method of applying a thermal stress to a living body with a free electron laser in the infrared region. We have been studying a method for introducing a gene using elastic expansion caused by the method. By irradiating the cell with a free electron laser (FEL) having a resonance wavelength in a cell membrane or the like, the gene can be efficiently, simply, and minimally invasively transferred to the cell. I found that it can be introduced. Furthermore, it has been found that by irradiating a continuously flowing cell line with a free electron laser beam, a gene can be selectively and continuously introduced into specific cells in a flowing phase. Therefore, the present invention provides a method for introducing a desired substance, such as a gene, into a specific cell, which can selectively and efficiently treat a large amount of cells by targeting individual cells. And an apparatus therefor.

[0008]

According to the present invention, a system comprising a cell and a substance such as a gene to be introduced into the cell is irradiated with a laser beam having an absorption wavelength of the cell, and the gene or the like is applied to the cell. And a device therefor. Further, the present invention provides a method for irradiating a cell and a system containing a substance to be introduced into the cell with a laser beam having an absorption wavelength of the cell to introduce the substance into the cell. The system containing the substance to be introduced is a mobile phase, and the system containing the cell and the substance to be introduced into the cell is continuously moved to the position irradiated with the laser light, and continuously. How to introduce the substance into the cells,
And a device therefor.

A free electron laser (hereinafter, referred to as FEL) having a wavelength that resonates with infrared absorption of a cell membrane or the like.
That. ), The cells, especially the cell membrane, absorb the laser light of that wavelength and are excited to cause elastic expansion due to thermal stress. Condition in which the elastic expansion caused by thermal stress when the FEL is irradiated to the subject, the time constant t _o, the light absorption coefficient mu _a, using a sound velocity v t _{o =} 1 / μ _a
v. When the pulse width of the FEL is shorter than t _o, the shock wave is propagated by stress gradient within the depth defined by 1 / mu _a, it is using the propagation distance z its strength,

[0010] _{σ (z) = Γμ a H} o exp (-μ a z)

Can be represented by Here, H _o is the energy density, gamma is a volume expansion coefficient g and heat capacity Cv gamma = g
v is a constant defined as ² / Cv. Effective impact is obtained by adjusting the wavelength of the FEL to a wavelength at which the absorption coefficient of interest is large.

FIG. 1 and FIG. 2 schematically show this state and will be described. FIG. 1 shows a state before the cells are irradiated with laser light, and FIG. 2 schematically shows a state of change when the cells are irradiated with laser light. 1 and 2 show plant cells as an example. In the case of animal cells, since there is no cell wall, only the cell membrane may be considered. Cell membranes are made up of countless lipids,
The cell wall is formed of a large number of celluloses and the like, and as shown in FIG. 1, these substances are usually closely associated with each other, and the extracellular substance P or P ′ cannot enter the cells. Can not.

However, as shown in FIG. 2, when the cells are irradiated with a laser beam having the absorption wavelength of the cell and the conditions shown by the above formula are satisfied, the laser beam is absorbed by the cells, especially the cell membrane and the cell wall. Then, elastic expansion occurs due to thermal stress. As a result, as shown in FIG. 2, the cell wall or the cell membrane instantaneously elastically expands, and a slight gap is formed in the portion of the cell wall or the cell membrane irradiated with the laser beam.
That is, a space such as a channel or a transporter is instantaneously generated in a cell membrane or a cell wall. However, unlike a channel or transporter, this space is completely physical, so that substances outside the cell can pass relatively freely inside and outside the cell. It is also conceivable that a laser beam hits a region which is also irradiated with a substance existing outside the cell, moves so that the substance is pushed by the light energy, and is pushed into the cell wall, a slight gap between cell membranes. .

The present inventors first examined whether or not a gene could be introduced into cells by this method. To this end, BY2 of tobacco leaf-derived cultured cells was used as a cell.
( Nicotiana tabacum L.) (5th and 12th days of culture) was used. The gene to be introduced is G that expresses a fluorescent substance.
FP gene to 35S promoter (35SC4PPDK)
Used with This gene was used by incorporating it into a plasmid vector. The plasmid incorporating the gene is referred to as a _"p35 -S GFP-TYG". The plasmid used was diluted to 1,0.5,0.1 mg / ml,
10 μl each for 100 μl of the concentrated BY2 cell suspension
l of the plasmid solution was added to prepare a sample. Therefore, the plasmid concentration of each sample was 100, 50,
It became 10 μg / ml. A plastic petri dish having a radius of about 3 cm was used as a reaction field, and a 100 μl sample was placed in a central recess of the petri dish, and laser was irradiated from an angle of 45 °.

FIG. 3 shows an outline of the apparatus used at this time. A sample 35 was placed at the center of a plastic Petri dish 37 and placed so that it could be observed with a microscope 36. On the other hand, a laser beam 30 was introduced from a hollow tube 31 and reflected by a mirror 32, and the sample 35 was irradiated with the laser beam at an angle of about 45 degrees through an FEL probe 33. The distance between the tip of the FEL probe 33 and the sample is about 3 to
It was made to be 5 cm. The characteristics of the FEL used at this time were: working wavelength: 5.75 μm average output: 12 mW irradiation range: 0.3 mmφ. Therefore, the output per unit area is 17.0 [W
/ Cm ² ]. The irradiation time was 1000 seconds.

After the irradiation, the cells were cultured at room temperature, and the expression of the GFP gene was confirmed 24 hours later. A filter for DAPI was used for observation of fluorescence by the GFP gene.
At this time, the fluorescence caused by dead cells was suppressed by Evans blue treatment during observation. The results are shown in Table 1 below.

Table 1 Sample name Culture days [days] DNA amount [ug / ml] Fluorescent cells [units] New10 5 10 170 New100 5 100 50 Old10 12 10 30 Old50 12 50 30 Old100 12 100 30 “New” in the table "" Means cells having been cultured for 5 days, and "O
"ld" is cells having 12 days of culture. The numerical value after each sample indicates the concentration of the plasmid in the sample.

The total number of cells observed was about 5,000,
From this, the introduction efficiency was a high value of 3 to 17%. The cell mortality at this time was about 5%. As for the difference in the transfection efficiency depending on the number of days of culture, it seems that the younger cells are still better, but the transfection efficiency of about 3% was obtained even with the cells of 12 days of culture. Regarding the concentration of the gene, it seems that the transfection efficiency tends to decrease when the gene concentration is excessively high.

The introduced GFP-TYG gene expresses a protein having fluorescence. When expressed, the GFP-TYG gene exhibits a fluorescence characteristic having a maximum excitation wavelength of 488 nm and a maximum emission wavelength of 507 nm. Therefore, when observing this, it is common sense to consider a FITC filter (excitation wavelength: 450-
490 nm, fluorescence wavelength: 515-565 nm should be more suitable, but because the signal was too weak to observe, the filter for DAPI (excitation wavelength: 360-370 nm, fluorescence wavelength: 4) was used in this experiment.
00 nm or more). Since the fluorescence characteristic of GFP has a wide tail, observation with a DAPI filter was also possible.

In order to observe the fluorescence due to the expression of the introduced gene, the image light of each sample irradiated with a laser beam (free electron laser) is applied to the CAPI through a DAPI filter.
The images were taken into a CD camera, imaged while checking on the monitor screen, and printed by a printer. FIGS. 6 to 15 show the obtained photograph and the photograph obtained by performing image processing on the photograph.
6 to 15 are photographs replacing the drawings.
FIG. 6 shows the results observed with a DAPI filter. The cells emit blue fluorescence in the background, weak red in dead cells, and green fluorescence in cells suspected of containing plasmid GFP. Were observed, but are represented in monochrome in the drawing. FIG. 7 is a color photograph showing the fluorescence observed when the DAPI filter is used, which is shown in monochrome in the drawing. Fig. 7 shows a photo scanned by a scanner. At the time of scanning, the difference between the background blue and the green fluorescence, which is thought to be caused by GFP, is almost indistinct. Identification by color is possible. FIG. 9 shows the result of the composition by the image processing. FIG. 8 is a color photograph showing the fluorescence observed when using the FITC filter, which is shown in monochrome in the drawing. FIG. 9 is a color photograph showing the result of the combination by the image processing, which is shown in monochrome in the drawing. 10 and 11 show images by visible light. FIG. 10 shows an image without FEL processing, and FIG. 11 shows an image after FEL processing. FIG.
2 and 13 show the results of fluorescence observation using a FITC filter. FIG. 12 shows the result after FEL treatment, and FIG. 13 shows the result after FEL treatment. FIG. 14 and FIG.
5 is a binarized image of each of FIGS. 12 and 13.

When observed with a DAPI filter, blue fluorescence was observed in the background, weak red in dead cells, and green fluorescence in cells suspected of containing GFP (see FIG. 6; the drawing is not color but monochrome). Is displayed only in.). In addition, for confirmation, observation was performed using a FITC filter (see FIG. 8; however, the drawing is displayed only in monochrome, not in color), but the fluorescence observed when the DAPI filter was used (see FIG. 7). The drawings are displayed only in monochrome, not in color.) And F
The positions of the fluorescence observed when using the ITC filter were almost the same. From this, it was confirmed that the use of the DAPI filter was effective. By the way, FIT
Even for cells having relatively strong fluorescence in which GFP expression can be clearly confirmed even with the C filter, the result was about 1/10 of the result obtained by DAPI observation.

The image processing for quantifying the difference from the background was performed as follows. Monochrome CC
Using a D-camera, an arbitrary area between the FEL-treated cells and the FEL-untreated cells was observed, and the fluorescence was compared. FIG. 10 (FEL unprocessed) and FIG.
(FEL processing). Next, the fluorescence was observed.
At the time of this observation, a FITC filter was used. This is because it was determined that the FITC filter was able to selectively pick up only the fluorescence considered to be caused by GFP. The results of the fluorescence observation are shown in FIG. 12 (FEL untreated) and FIG.
L processing) (By the way, the integration time is 90 seconds in both cases.) The result of binarizing the obtained image is shown in FIG.
(FEL unprocessed) and FIG. 15 (FEL processed). In each figure, the area of the region remaining white was compared. As a result, FEL unprocessed: FEL processed = 4: 2287 (unit is the number of dots), and a clear difference was confirmed.

In this experiment, FITC, D
Observations were made using two API filters. As a result, when observing with the naked eye, the use of the DAPI filter increases the G value more than the use of the FITC filter.
It was shown that the fluorescence attributed to FP could be observed. Also, with the FITC filter, weak fluorescence could be observed by increasing the integration time at the time of imaging, and the results showed that there was a clear difference from the control sample. As a result, the FEL of the present invention
It was found that the GFP gene could be introduced into the plant cells by the treatment.

As is clear from the above experimental results, the system in which the cell and the gene to be introduced are present is:
By irradiating a laser beam having an absorption wavelength of a cell, a gene can be introduced from a gap of a cell membrane expanded by a shock wave due to energy of the absorbed laser beam, and the present invention provides a simple and highly efficient gene transfer method. Is what you do.

In the above description, the gene transfer method by bulk (batch type) has been described. Next, the continuous gene transfer method of the present invention will be described with reference to the drawings. FIG.
1 is an overall schematic configuration diagram of a continuous gene introduction device of the present invention. FIG. 5 is a cross-sectional view of the substrate 1 taken along the line BB. The substrate 1 is provided with a groove 2 through which a cell C and a system M containing a gene to be introduced can move in a certain direction. A light transmitting material 3 is provided on the groove 2 of the substrate 1. In the illustrated example, the light transmitting material 3 is provided over the entire length of the substrate 1. However, the light transmitting portion is provided only partially to cover the laser light irradiation area, and the groove 2 is provided before and after the portion. May be formed of a metal or plastic plate having no light transmissivity, and the light transmitting material 3 may be provided only in the laser light irradiation area, or an opening through which the laser light can be irradiated depending on the sample. . Further, as a material of the light transmitting plate 3, an optical element plate having a small absorption with respect to the wavelength of the irradiation laser light is used, and for example, a material made of BaF, KBr, or diamond having a small absorption with respect to the infrared light is used. . Irradiation laser light may be of an absorption wavelength of a cell into which a gene is to be introduced, and preferably has a wavelength in an infrared region.

At the front and rear ends of the substrate 1, a cell M and a system M containing a gene to be introduced into the groove 2 are supplied.
Connecting member 4 for discharging the laser beam after irradiation,
Transport pipes 5, 5 ′ are connected via 4, and a system 6 containing cells C and a gene to be introduced is sent from a pump 6 to an infusion pipe 5 on the upstream side. Although not shown in the figure, a concentrated cell suspension obtained by adding a gene solution to the system M is added to an infusion tube along the route, mixed and sent. The mixed solution containing the cell suspension into which the gene has been introduced is sent out by the transport pipe 5 ′, and
Is stored in the storage unit 7 after being irradiated. The system M containing the cells C sent from the pump 6 and the gene to be introduced is continuously transported to the groove 2 via the transport pipe 5 and the connecting member 4. Then, when reaching the predetermined position of the groove 2, the laser light L is irradiated through the light transmitting material 3, and the gene is introduced into the cell C. The cells into which the gene has been introduced further move, and are stored in the storage unit 7 via the connecting member 4 and the transport pipe 5 '. The laser light L emitted through the light transmitting material 3 is obtained by reducing or enlarging the laser light sent from the laser generator 10 through the optical fiber 11 and reducing or enlarging it by an appropriate lens (convex or concave) means. The laser beam having a predetermined diameter is sent through the channel 12 and is irradiated to the cells C of the mixed solution M flowing in the groove 2.

As described above, the continuous gene introduction apparatus of the present invention can continuously and automatically introduce a desired gene into many cells by irradiating a laser beam. The conventional gene transfer apparatus is of a batch type and requires operation in each batch, which is complicated, but the continuous gene transfer apparatus of the present invention is characterized in that it can be performed continuously and automatically. Things. Also, if a marker that can be automatically sorted by a cell sorter or the like is attached to the gene to be introduced, the cell into which the gene is introduced is sorted by placing the cell sorter at the location of the transport tube 5 ', and the gene is not introduced. By circulating the cells through the transport tube 5, complete automation can be performed. In the above-described continuous gene transfer apparatus of the present invention, gene transfer has been described. However, low molecular substances such as proteins and drugs can be used instead of genes.

[0028]

DETAILED DESCRIPTION OF THE INVENTION As described above, the present invention introduces a gene into a cell by irradiating a system containing a cell and a gene to be introduced into the cell with laser light having an absorption wavelength of the cell. A method and an apparatus therefor are provided. The cells in this method of the present invention may be plant cells having a cell wall or animal cells without a cell wall. It may also be a cell of a microorganism. There is no particular limitation as long as the cell membrane is irradiated with a specific laser beam and elastic expansion occurs due to thermal stress, but some cells are weak to thermal stress due to laser beam,
Before transfection, it is also necessary to examine the degree of resistance to thermal stress in advance. The cells to which the method of the present invention can be applied are, for example, not limited to the cigarettes of plants shown in the above examples, and rice, potatoes, soybeans, plant cells such as corn, humans, rats, mice, hamsters, rabbits, Examples include animal cells such as dogs and microbial cells such as yeast and Escherichia coli.

Although a single cell is preferably applied to the method of the present invention, various cells may be mixed. Since cells have characteristic absorption wavelengths corresponding to each cell, even if various cells are mixed, the gene can be selectively introduced only into the target cells by specifying the wavelength of the laser beam to be irradiated. Is also possible. Even in the same individual, the absorption wavelength may differ depending on the cells of each organ.Therefore, even in a system in which various types of cells are mixed, the target gene can be selected only for specific cells by selecting the wavelength of the laser beam to be irradiated. One of the characteristics of the method of the present invention is that it can be introduced.

The gene to be introduced may be a short gene such as a probe or antisense, or may be a structural gene as described in the above example or a plasmid vector containing the same. In addition, the gene may be DNA or RNA.
DNA or RNA may be single-stranded or double-stranded. According to the method of the present invention, it is possible to reach the nucleus of a cell, so that the introduced gene does not necessarily need to be incorporated into a vector. When a gene is to be introduced into a genome, a conventional method can be used. For example, a gene having a base sequence required for homologous recombination added to both ends or in the vicinity of a gene to be introduced can also be used. When a gene is introduced into a prokaryotic cell such as Escherichia coli, the method of the present invention can be carried out simply and efficiently.

As the laser beam, a wavelength at which elastic expansion is caused only by target cells due to thermal stress may be selected.
Therefore, it is preferable that the range of the wavelength of the generated laser beam is extremely narrow, and that the laser of a specific wavelength can be continuously generated because the characteristic absorption wavelength differs depending on the cell. The laser source may be a semiconductor laser device or another laser device, but the wavelength range of the generated laser light is narrow, and a free electron laser capable of continuously changing the wavelength is the present invention. Preferred for the method. Also, when examining the characteristic absorption wavelength of various cells, laser light from a free electron laser device is convenient. In the cell where the gene is to be introduced,
By continuously irradiating the laser beam from the free electron laser device at a different wavelength, the absorption spectrum of the cell can be measured. For example, FIG. 16 shows an absorption spectrum of tobacco leaf cells used in the above-described experiment. By taking such a spectrum, it can be seen that the maximum absorption is at about 5.75 μm in tobacco leaf cells. Therefore, it can be seen that the gene can be introduced by the method of the present invention by irradiating the cells with 5.75 μm laser light. As a specific example of the wavelength of the laser beam of the present invention, an infrared region for causing cells to undergo elastic expansion due to thermal stress is preferable, and more specifically, for example, the wavelength is 1 to 20 μm, preferably 3 to 15 μm. , 3 to 1
It is about 0 μm.

The laser beam irradiation method of the present invention includes
Although continuous irradiation or pulsed irradiation may be performed, pulsed irradiation is preferable because an impact is given. When irradiating a laser beam in a batch system such as a bulk system, an appropriate pulse frequency can be selected.However, in the continuous gene transfer method of the present invention, the moving speed of the system containing the gene and the pulse rate are controlled. frequency,
It is important to adjust the irradiation frequency. The pulse frequency and the output of the laser beam are adjusted in order to irradiate the pulse a required number of times during the time when the laser beam can move within the irradiation range of the laser beam. The pulse frequency is usually 5 to 4000 times / second, preferably 10 to 3500 times / second.
Seconds, or about 10 to 3330 times / second. There is no particular limitation on the output of the laser beam, but if it is too strong, the mortality of the cells will increase, and if it is too weak, it will not produce a sufficient effect, so it is necessary to select an efficient output according to the type of cell There is.

The present invention also provides a method for introducing a substance into a cell by irradiating a system containing the cell and a substance to be introduced into the cell with a laser beam having an absorption wavelength of the cell. The system containing the substance to be introduced into the cells is a mobile phase, and the system containing the cells and the substance to be introduced into the cells is continuously moved to the position irradiated with the laser light, A method of continuously introducing a substance into cells,
And an apparatus therefor. The cells and laser light in the method of the present invention are as described above. Substances to be introduced into cells in the continuous method of the present invention are not limited to the genes exemplified above, but include proteins and drugs. Proteins, drugs, and the like can be introduced into specific cells in the same manner as the aforementioned genes. The protein may be any protein having a certain function in or on a target cell, and may be not only an enzyme, an antibody, a cytokine, various factors, but also a receptor immobilized on a cell membrane in some cases. You may. In addition, as the drug, those used as active ingredients of pharmaceuticals and bioactive substances thereof can be used.

The system containing the cells and the substance to be introduced into the cells in the method of the present invention is preferably in the form of a solution, but is not limited thereto. It may be a suspension or, depending on the case, a fluid having low fluidity. For example, even in the case of a gel, the laser light L is moved with respect to the gel fixed to the groove 2 shown in FIG. The system may be able to move in a certain direction. Therefore, when the cells are fixed, the method of the present invention is such that the laser beam L moves, so that the cells and the system containing the substance introduced into the cells can move in a certain direction. It also includes the method of becoming.
For example, when a substance such as a gene is to be introduced into cells of a blood vessel wall, a method of irradiating by moving a laser beam while contacting a solution containing the substance to be introduced into the blood vessel wall is also included. Things.

As described above, the method of the present invention can also be used in combination with a cell sorting device such as a cell sorter. The cell sorting device may be provided before irradiation with laser light, or may be provided after irradiation,
You may provide in both. For example, it is possible to provide such a sorting device before irradiation with laser light, and to send out only the selected cells to the laser light irradiation device, or to provide such a sorting device after irradiation with laser light, and a substance is provided. It is also possible to sort only the introduced cells. In addition, cells into which the substance has not been introduced can be recycled. Also, both can be used in combination. The method of the present invention can also be directly or indirectly connected with a living body like the current kidney dialysis. For example, it is also possible to adopt a method in which blood is taken out, firstly separated into only plasma, a specific cell in plasma is targeted, the method of the present invention is applied, and then circulated again to the living body. Therefore, the method of the present invention includes not only a method applied to a cell line removed from a living body, but also a method or an apparatus for recycling the same to a living body.

The device of the present invention may be any device capable of performing the above-described method of the present invention, and its shape, size, material and the like can be appropriately designed.

[0037]

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 Preparation of Samples Tobacco plant cells BY2 cells were suspended in MS medium, and each 100 μl of concentrated BY2 cell suspension was prepared in Example 2
10 μl of the plasmid solution prepared in 1) was added to prepare a sample. Therefore, the plasmid concentration of each sample was 10
0, 50 and 10 μg / ml.

Example 2 Introduction of Gene Using the apparatus shown in FIG. 3, each sample was placed in a central cavity of a plastic petri dish having a radius of about 3 cm to form a reaction field, and irradiated with laser light from an angle of 45 degrees. The laser beam used was a laser beam from a free electron laser device. Working wavelength: 5.75 μm Average output: 12 mW Irradiation range: 0.3 mmφ Output per unit area: 17.0 W / cm ² Irradiation time: 1000 seconds The tobacco plant cell BY2 used as a sample was a laser beam having the above-mentioned wavelength. The maximum absorption wavelength is specified by analyzing the results of the absorption wavelength (see FIG. 16) obtained by separately irradiating the free electron laser light with various wavelengths. After irradiation with laser light, the cells were cultured at room temperature as they were. The expression of plasmid GFP was confirmed 24 hours later. The results are shown in Table 1 above.

[0040]

As described in detail above, in the method and apparatus for gene transfer according to the present invention, a mixture containing moving cells and a gene is irradiated with a laser beam having the maximum absorption wavelength on the cell membrane, and a stress gradient is generated by the shock wave. A slight gap is created in the cell membrane due to elastic expansion that occurs instantaneously from, and the gene is introduced through the gap, so different types by irradiating laser light of a specific wavelength that creates a gap due to the elastic expansion of the cell membrane The advantage is that the gene can be introduced selectively and efficiently into the type of cells that selectively absorbs the laser beam of the specific wavelength at the maximum even when the cells exist.

[Brief description of the drawings]

FIG. 1 is an overall schematic configuration diagram of a gene introduction device.

FIG. 2 is an explanatory diagram of a gene introduction method.

FIG. 3 is an absorption wavelength spectrum chart of a cell.

FIG. 4 is an overall schematic configuration diagram of an example of a continuous gene introduction device of the present invention.

FIG. 5 is a cross-sectional view taken along the plane BB of the substrate 1 in the overall schematic configuration diagram of the continuous gene transfer apparatus of the present invention.

FIG. 6 is a photograph instead of a drawing, showing the result of observing the result of gene transfer by the method of the present invention using a DAPI filter.

FIG. 7 is a color photograph showing fluorescence observed when a DAPI filter is used as a result of gene transfer according to the method of the present invention. In this drawing, a photograph replacing the drawing shown in black and white is shown. .

FIG. 8 is a color photograph showing fluorescence observed when a FITC filter is used as a result of gene transfer according to the method of the present invention. In this drawing, this photograph is a photograph replacing a drawing shown in monochrome. .

FIG. 9 is a color photograph showing the result of synthesis by image processing of the result of gene transfer according to the method of the present invention, but is a photograph replacing the drawing shown in black and white in this drawing.

FIG. 10 shows the result of gene transfer according to the method of the present invention as an image by visible light, and is a photograph replacing a drawing in the case where FEL is not processed.

FIG. 11 shows the result of gene transfer according to the method of the present invention in the form of an image using visible light, and is a photograph replacing the drawing after FEL processing.

FIG. 12 shows the result of fluorescence observation using a FITC filter on the result of gene transfer according to the method of the present invention, and is a photograph replacing a drawing in the case of no FEL treatment.

FIG. 13 shows the result of fluorescence observation using a FITC filter on the result of gene transfer according to the method of the present invention, and is a photograph replacing the drawing after FEL treatment.

FIG. 14 is a photograph replacing a drawing when the image of FIG. 12 showing the result of gene transfer by the method of the present invention is binarized.

FIG. 15 is a photograph instead of a drawing when the image of FIG. 13 is binarized and shows the result of gene transfer by the method of the present invention.

FIG. 16 is a chart showing measured absorption spectra of cells of tobacco leaves used in the experiment of the present invention. As shown in FIG. 16, it can be seen that in the cells of the tobacco leaves, the maximum absorption is at about 5.75 μm.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Introduction plate 2 Groove 3 Light transmission plate 4 Connection member 5-5 'Infusion tube 6 Pump 7 Storage part 10 Laser generator 11 Optical fiber C cell M System containing gene to be introduced L Laser light 20 Cell 21 Nucleus 22 Cell membrane 23 Cell Wall 30 Laser Light (FEL) 31 Hollow Fiber 32 Mirror 33 Laser Light Irradiation Port 35 Sample 36 Observation Table 37 Petri dish

Claims (21)

[Claims] 1. A method for introducing a gene into a cell by irradiating a system containing a cell and a gene to be introduced into the cell with laser light having an absorption wavelength of the cell. 2. The method according to claim 1, wherein the absorption wavelength of the cell is in the infrared region. 3. The method according to claim 1, wherein the laser light is from a free electron laser. 4. The laser beam according to claim 1, wherein the laser beam is pulsed.
A method according to any one of claims 1 to 3. 5. The method of claim 4, wherein the number of pulses is between 5 and 3500 times per second. 6. The method according to claim 1, wherein the gene is DNA. 7. The method according to claim 1, wherein the gene is a gene integrated into a vector. 8. A method for irradiating a system containing a cell and a substance to be introduced into the cell with a laser beam having an absorption wavelength of the cell to introduce the substance into the cell, the method comprising introducing the cell and the substance into the cell. The system containing the substance to be introduced is the mobile phase, and the system containing the cells and the substance to be introduced into the cells is continuously moved to the position irradiated with the laser light, and the cells are continuously How to introduce substances into 9. The method according to claim 8, wherein the absorption wavelength of the cell is in the infrared region. 10. The method according to claim 8, wherein the laser light is from a free electron laser. 11. The method according to claim 8, wherein the laser light is pulsed. 12. The method of claim 11, wherein the number of pulses is between 5 and 3500 times per second. 13. The method according to claim 8, wherein the substance is a gene, a protein, or a drug. 14. The method according to claim 8, wherein the system comprising the cells and the substance to be introduced into the cells is in the form of a solution. 15. The moving speed of the mobile phase is adjusted based on the pulse irradiation speed (times / second).
The method according to any of the above. 16. A device for holding a cell and a system containing a gene to be introduced into the cell, and a laser device, wherein the laser device continuously or pulsed laser light having an absorption wavelength of the cell. A gene transfer device configured to irradiate and transfer a gene into cells. 17. A device comprising a groove capable of moving a cell and a system containing a substance to be introduced into the cell in a predetermined direction, and a laser device capable of irradiating a predetermined position of the groove with laser light. A device for introducing substances into cells. 18. The apparatus according to claim 16, wherein the laser light is pulsed. 19. The apparatus according to claim 17, wherein the groove capable of moving the system in a certain direction is provided with an introduction plate made of a light transmitting member. 20. The device according to claim 17, wherein the substance is a gene, a protein, or a drug. 21. The apparatus according to claim 17, wherein the system containing the cells and the substance to be introduced into the cells is in the form of a solution.