JP2008136400A - Gene transfer device and method therefor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a gene transfer device which can transfer genes at high transfer efficiency, with minimal invasion, while maintaining high survival rate, and to provide a method therefor. <P>SOLUTION: This gene transfer device 10 for transferring genes into a cell 70 comprises an transfer needle 44 that perforates the cell membrane 72 of a cell 70, cultured in a DNA-dispersed solution 66 in which the genes to be transfer is dispersed, and a driving unit 48 and a Z-axis actuator 56 that makes perforated, at at least two positions of the cell membrane 72. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a gene introduction apparatus and method for introducing a gene into a cell.

In Patent Document 1, a substance such as a gene is electrically adsorbed on the tip of a fine needle, the fine needle is allowed to enter the cell, and a pulse voltage is applied to the fine needle to introduce the substance into the cell. A microinjection method and apparatus for introducing the above are disclosed. Here, the penetration of the fine needle is performed by fine movement using a piezoelectric element that expands and contracts coaxially with the fine needle.
WO04 / 092369

  The above-mentioned Patent Document 1 is a method of holding a gene at the tip of a fine needle, can introduce a gene with minimal invasiveness to cells, and can obtain a high survival rate.

  However, since the surface area for retaining genes is very small, the amount of genes retained is very small, and it is difficult to introduce an effective amount of genes into cells. For this reason, it is difficult to obtain high introduction efficiency.

  The present invention has been made in view of the above points, and an object of the present invention is to provide a gene introduction apparatus and method having high introduction efficiency while maintaining a high survival rate with low invasiveness.

One aspect of the gene introduction device of the present invention is a gene introduction device for introducing a gene into a cell.
A perforation means for perforating a cell membrane of a cell cultured in a culture solution in which the gene to be introduced is dispersed;
Driving means for driving the perforation means to perforate at least two locations of the cell membrane;
It is characterized by comprising.

In addition, one aspect of the gene introduction method of the present invention is:
Selecting a cell into which the gene is to be introduced;
Perforating at least two portions of the cell membrane of the cell;
It is characterized by having.

  According to the present invention, a gene is dispersed in an extracellular solution, and pores are formed in at least two locations of the cell membrane. It is possible to provide a gene transfer apparatus and method that have a high transfer efficiency and high transfer efficiency.

  The best mode for carrying out the present invention will be described below with reference to the drawings.

[First Embodiment]
First, a gene introduction device and a gene introduction method according to a first embodiment of the present invention will be described with reference to FIGS. 1 to 3C.

  As shown in FIG. 1, the gene introduction apparatus 10 according to the present embodiment is provided in an inverted microscope 12 for observing cells.

  The inverted microscope 12 includes a work holder 16 on which a work 14 containing cells is placed, an illumination device 18 that illuminates the cells on the work holder 16, light reflected or transmitted through the cells, or fluorescence generated from the cells. A microscope XY stage 22 that moves the work holder 16 in the X and Y directions, a microscope XY stage controller 24 that drives and controls the microscope XY stage 22, the illumination device 18 and the observation device 20, a microscope controller 26 that controls the image 20, an image processing device 28 that processes the image obtained by the observation device 20, and a monitor 30 that displays the image processed by the image processing device 28. And a control computer 32 for controlling the entire inverted microscope 12.

  In addition, the illumination device 18 collects the illumination light emitted from the transmitted illumination light source 34 on the cell and the transmitted illumination light source 34 that irradiates the cell with illumination light from the side opposite to the observation device 20. And an epi-illumination light source 38 that irradiates the cells with illumination light from the same direction as the observation device 20.

  On the other hand, the observation apparatus 20 includes an observation optical system including an objective lens (not shown), a CCD camera 40 that captures light from the cell via the observation optical system and acquires an image, and direct light from the cell. An eyepiece 42 for observation is provided.

  The gene introduction device 10 according to the present embodiment is arranged in the vicinity of the work holder 16 of the inverted microscope 12, and the introduction needle 44 inserted into the cell and the introduction needle holder for holding the introduction needle 44. 46, a drive unit 48 that moves the introduction needle 44 by moving the introduction needle holder 46, and a control box 50 that controls the gene introduction apparatus 10. Here, the drive unit 48 includes, for example, a three-axis linear movement mechanism that moves the introduction needle 44 in the X, Y, and Z directions.

  As shown in FIG. 2A, the introduction needle 44 is provided on the free end side of the cantilever 52 so as to extend in a direction intersecting the longitudinal direction of the cantilever 52. The cantilever 52 is fixed to an attachment member 54, and the attachment member 54 is fixed to an arm 46 </ b> A of the introduction needle holder 46 via a Z-axis actuator 56. As a result, the cantilever 52 is held obliquely downward, and the introduction needle 44 is held at the free end of the cantilever 52 with its tip directed vertically downward.

  As shown in FIG. 2B, the cantilever 52 includes a flexible silicon base portion 58 and a lever portion 60 in which a fine introduction needle 44 is formed at the tip. It is fixed to be exchangeable. Therefore, by exchanging the cantilever 52, the gene introduction apparatus 10 can be used repeatedly without fear of contamination.

  The Z-axis actuator 56 disposed between the mounting member 54 and the introduction needle holder 46 is composed of, for example, stacked piezoelectric elements, and expands and contracts according to the applied voltage. As shown in FIG. 2A, since the Z-axis actuator 56 is disposed vertically between the mounting member 54 and the introduction needle holder 46, the applied voltage is adjusted so that the introduction needle holder 46 is controlled. Thus, the attachment member 54 is slightly displaced in the vertical direction, and the tip of the introduction needle 44 attached vertically downward to the attachment member 54 can be moved by a minute distance in the vertical direction.

  The control box 50 includes a drive unit control circuit 62 that controls the drive unit 48 and an actuator control circuit 64 that controls the Z-axis actuator 56.

  A gene introduction method using the gene introduction apparatus 10 according to the present embodiment configured as described above will be described below.

  In order to introduce a gene into cells cultured in the culture medium in the workpiece 14 using the gene introduction apparatus 10 according to this embodiment, first, the gene introduction substance is dispersed in the cell culture medium. Then, the microscope XY stage controller 24 and the microscope controller 26 are operated, and the cells to be observed by the operation of the microscope XY stage 22 are arranged under the microscope observation (details will be described in a third embodiment described later). Thereafter, the drive unit 48 is operated by the operation of the drive unit control circuit 62, and the introduction needle 44 of the cantilever 52 is brought close to the cell from above the cell. Since the cells seeded on the substrate 68 of the workpiece 14 have only a thickness of about 2 μm to 10 μm, the minute descent from this close position is caused by operating the Z-axis actuator 56 by the operation of the actuator control circuit 64. Do.

  By operating the Z-axis actuator 56, the introduction needle 44 is lowered and brought closer to the substrate 68 as shown in FIG. Thereby, in the middle of the tip of the introduction needle 44 descending, it enters the culture solution in which the gene in the workpiece 14 is dispersed (hereinafter referred to as DNA dispersion solution 66) and is seeded on the substrate 68 of the workpiece 14. Contact the cell 70. Here, the introduction needle 44 is further lowered to perforate the cell membrane 72 and the nucleus 74.

  Then, as shown in FIG. 3B, if the introduction needle 44 is lowered to a position where the tip of the introduction needle 44 comes into contact with the surface of the substrate 68, the introduction needle 44 is moved further than the contact position with the surface of the substrate 68. Lower. As a result, as shown in FIG. 3C, the tip of the introduction needle 44 slides with respect to the surface of the substrate 68, and by this sliding, the bottom surface side is also broken, and 2 on the top surface side and bottom surface side of the cell membrane 72. A hole penetrating between the broken portions 76 is opened.

  When the DNA dispersion solution 66 flows through the thus formed through-hole, the gene dispersed in the DNA dispersion solution 66 flows into the cell 70.

  Note that after the introduction needle 44 is raised and the introduction needle 44 is pulled out of the cell 70, the cell membrane 72 is recovered by self-repair and a gene is taken into the cell 70 after a certain period of time.

  In this way, in this embodiment, the gene can be introduced into the cell 70 with certainty and high introduction efficiency, with the minimally invasive and high survival rate as in the prior art.

  Note that the sliding method of the introduction needle 44 is not limited to the method of lowering the introduction needle 44 further than the contact position with the surface of the substrate 68 as described above. For example, the introduction needle holder 46 is used. The arm 46A is also composed of stacked piezoelectric elements in the same manner as the Z-axis actuator 56. By extending and contracting the arm 46A, the introduction needle 44 is moved horizontally with the tip of the introduction needle 44 in contact with the substrate 68. Can also be done.

  Of course, when the drive unit 48 can finely adjust in the XYZ directions, it is not necessary to configure the Z-axis actuator 56 and the arm 46A with piezoelectric elements.

[Example 1]
FIG. 4A is an example in which Hela S3 cells are immersed in a fluorescent dye solution and observed with a laser scanning microscope. A horizontal cross-sectional image of cells 2 to 3 μm above the substrate surface to which the cells are attached Become. As the fluorescent dye, sulforhodamine is used. In the laser scanning microscope, in order to observe this fluorescent image, the solution portion around the cell is observed.

  In Example 1, after the introduction needle 44 was lowered and entered the cell, the cell was further lowered from the position where the tip of the introduction needle 44 was in contact with the surface of the substrate 68. As a result, the tip of the introduction needle 44 is slid by about 3 μm on the surface of the substrate 68, two holes are made in the cell membrane, and an attempt is made to introduce the dye solution into the cell. FIG. 4B is a diagram showing this result. Since the dye solution around the cells flows into the perforated cells, the fluorescence intensity in the cells increases and it can be confirmed that the cells are taken into the cells.

[Example 2]
As in Example 1, an example in which HelaS3 cells are immersed in a gene solution and introduction is attempted is shown. The introduced gene is a gene that expresses a GFP fluorescent protein, and the correctness of the introduction can be confirmed by fluorescence observation.

  FIG. 5 (A) shows a microscopic observation image of a cell which was attempted to be introduced immediately after gene introduction. We are trying to select and introduce multiple cells in the observation image. FIGS. 5B and 5C are microscopic observation images in which the success or failure of the introduction was confirmed after 24 hours from the introduction. FIG. 5B is a phase difference observation image and shows the state of the cells after 24 hours. FIG. 5C shows the cells observed by fluorescence observation. In the cells that have been successfully introduced, the gene is expressed and strong fluorescence intensity is obtained. From this result, it can be confirmed that the gene is introduced into the cell very efficiently.

[Second Embodiment]
Next, a gene introduction device and a gene introduction method according to a second embodiment of the present invention will be described with reference to FIGS.

  In the present embodiment, a cantilever 52 as shown in FIG. 6 is used. That is, the cantilever 52 in this embodiment includes a flexible silicon base portion 58, a lever portion 601 in which a fine introduction needle 441 is formed at the tip, and a lever in which a fine introduction needle 442 is also formed at the tip. (In FIG. 6, the introduction needles 441 and 442 are shown extending in a horizontal direction with respect to the lever portions 601 and 602, but actually, in the first embodiment described above, FIG. Like the introduction needle 44 and the lever portion 60, the introduction needles 441 and 442 are provided so as to extend in a direction intersecting with the extending direction of the lever portions 601 and 602).

  A gene introduction method using the gene introduction apparatus 10 according to the present embodiment configured as described above will be described below.

  In order to introduce a gene into the cells 70 cultured in the culture medium in the workpiece 14 using the gene introduction apparatus 10 according to this embodiment, first, the gene introduction substance is dispersed in the cell culture medium. Then, the microscope XY stage controller 24 and the microscope controller 26 are operated, and the cells to be observed by the operation of the microscope XY stage 22 are arranged under the microscope observation. Thereafter, the drive unit 48 is operated by the operation of the drive unit control circuit 62, and the introduction needles 441 and 442 of the cantilever 52 are brought close to the cell from above the cell.

  At this time, the introduction needle 44 is lowered by operating the Z-axis actuator 56 by the operation of the actuator control circuit 64. As a result, while the tips of the introduction needles 441 and 442 are descending, they enter the DNA dispersion solution 66 in the workpiece 14 and come into contact with the cells 70 seeded on the substrate 68 of the workpiece 14. Here, as shown in FIG. 7, the introduction needles 441 and 442 are further lowered to perforate the cell membrane 72 and the nucleus 74.

  As a result, as shown in FIG. 8, two holes 78 can be simultaneously formed in the cell membrane 72 on the upper surface of the cell 70 by the two introduction needles 441 and 442 from the outside of the cell 70. As the DNA dispersion solution 66 flows through the thus formed through-hole, the gene dispersed in the DNA dispersion solution 66 flows into the cell 70.

  In addition, after raising the introduction needles 441 and 442 and withdrawing the introduction needles 441 and 442 from the cell 70, the cell membrane 72 recovered by self-repair and a gene was taken into the cell 70 after a certain period of time. It becomes a state.

  According to the second embodiment described above, even if the cell has a weak adhesive force with the substrate 68, the cell 70 is detached from the substrate 68 by sliding the fine introduction needle with respect to the substrate 68. It is possible to introduce the gene reliably.

  Even in the state where the cells 70 are stacked, it is possible to select only the cells on the upper side and introduce the gene.

  In addition, by punching the cell membrane 72 twice using the cantilever 52 using the single introduction needle 44 as described in the first embodiment, the two holes are perforated before the cell membrane 72 is self-repaired. Of course, 78 may be performed.

[Third Embodiment]
Next, a gene introduction device and a gene introduction method according to a third embodiment of the present invention will be described with reference to FIGS.

  As shown in FIG. 9, a laser pointer 82 that emits a weak output laser beam and a laser beam emitted from the laser pointer 82 in the frame 80 (see FIG. 1) of the inverted microscope 12 A half mirror 84 that reflects toward the right, a half mirror 86 that reflects laser light reflected by the half mirror 84 in the right direction in the figure, and a laser beam reflected by the half mirror 86 toward the upper side in the figure. A reflecting mirror 88, an objective lens 90 that condenses the laser light reflected by the mirror 88, and an actuator 92 for adjusting the focal position of the objective lens 90 are provided. The substrate 68 of the work 14 is formed of a transparent member such as glass. The driving of the laser pointer 82 and the actuator 92 is controlled by the control computer 32 via a corresponding controller, although not specifically shown.

  With this configuration, spot light is formed at the cell 70 position, and the image is captured by the CCD camera 40 via the objective lens 90, the mirror 88, and the half mirror 86. Cells 70 and spot positions 94 as shown are displayed. In the first and second embodiments, the microscope XY stage controller 24 and the microscope controller 26 are operated, and by operating the microscope XY stage 22, the cell 70 selected as a gene introduction target is aligned with the spot position 94, Two portions of the cell membrane 72 of the cell 70 are perforated by the introduction needle 44 or 441,442.

  On the other hand, the gene introduction apparatus 10 according to the third embodiment has a high output laser light source 96 as shown in FIG. 9 instead of the introduction needle 44 as in the first and second embodiments. A mirror 98, the laser beam from the laser light source 96 is reflected by the mirror 98 toward the half mirror 84, collected by the objective lens 90 through the half mirrors 84 and 86, and the mirror 88, and then applied to the cell 70. It is configured to irradiate. The laser light from the laser pointer 82 and the laser light from the laser light source 96 are calibrated in advance so that their laser optical axes coincide with each other. The drive of the laser light source 96 is controlled by the control computer 32 via a corresponding controller, although not specifically shown.

  A gene introduction method using the gene introduction apparatus 10 according to the present embodiment configured as described above will be described below with reference to the flowchart of FIG.

  In order to introduce a gene into the cells 70 cultured in the culture medium in the workpiece 14 using the gene introduction apparatus 10 according to this embodiment, first, the gene introduction substance is dispersed in the cell culture medium. Then, the cell 70 to which the gene is to be introduced is selected (step S10), and the selected cell 70 is moved to the spot position 94 (step S12).

  Thereafter, as shown by arrows in FIG. 9, the objective lens 90 is vibrated in the vertical direction of the cell 70 by the actuator 92 (step S14). The amplitude of the vibration at this time is set to be sufficiently higher than the known average cell 70 height. That is, it is vibrated at a position where the focal position penetrates the bottom surface and the top surface of the cell 70. For example, it is a position where the cell 70 becomes a blurred image on the upper surface side under microscopic observation.

  Then, at the time when holes are formed in the cell membrane 72, laser light is irradiated from the laser light source 96 to perforate at least two portions of the cell membrane 72 (step S16). Thereafter, the driving of the actuator 92 is stopped (step S18), and the process ends.

  In this way, laser light is irradiated through the top and bottom surfaces of the cell membrane 72, and the cell membranes 72 on both sides are perforated to form through holes. When the DNA dispersion solution 66 flows through the through-hole thus formed, the gene dispersed in the DNA dispersion solution 66 flows into the cell 70. The cell membrane 72 is recovered by self-repair and the gene is incorporated into the cell 70.

  As described above, according to the third embodiment, since drilling is performed with laser light, drilling can be performed at high speed. In particular, since the laser beam is irradiated while the objective lens 90 is vibrated at high speed, the upper surface and the bottom surface of the cell membrane 72 can be uniformly irradiated with the laser beam. Can be formed.

  Further, since maintenance such as replacement of an introduction needle that contacts the cell 70 and sterilization is unnecessary, it is easy to maintain performance.

  Note that the laser light source 96 may be irradiated with laser light intermittently in conjunction with the operation of the actuator 92 so as to perforate only two places on the upper surface and the bottom surface of the cell membrane 72.

  Further, the actuator 92 is used to puncture the upper surface of the cell membrane 72 by aligning the focal position of the laser beam with the upper surface of the cell membrane 72, and then puncture the bottom surface of the cell membrane 72 by aligning the focal position of the laser beam with the bottom surface of the cell membrane 72. In this way, drilling may be performed in time series.

  Although the present invention has been described above based on the embodiments, the present invention is not limited to the above-described embodiments, and various modifications and applications are naturally possible within the scope of the gist of the present invention.

  For example, the substance to be introduced into the cell is not limited to a gene, but may be any substance that can be turbid in a solution, such as a dye, a fluorescent reagent such as a quantum dot, an ion, a peptide, a protein, or a polysaccharide.

  Moreover, although the said embodiment shall perforate two places of a cell, of course, you may perforate more places.

(Appendix)
The invention having the following configuration can be extracted from the specific embodiment.

(1) In a gene introduction apparatus for introducing a gene into a cell,
A perforation means for perforating a cell membrane of a cell cultured in a culture solution in which the gene to be introduced is dispersed;
Driving means for driving the perforation means to perforate at least two locations of the cell membrane;
A gene introduction device comprising:

(Corresponding embodiment)
The first to third embodiments correspond to the embodiment related to the gene introduction device described in (1). In these embodiments, the cell 70 is the cell, the gene transfer device 10 is the gene transfer device, the DNA dispersion solution 66 is the culture medium in which the gene to be introduced is dispersed, the cell membrane 72 is the cell membrane, the cantilever The introduction needle 44 or 441, 442 provided in 52, or the laser light source 96 and the objective lens 90 correspond to the punching means, and the drive unit 48 and the Z-axis actuator 56 or the actuator 92 correspond to the drive means.

(Function and effect)
According to the gene introduction device described in (1), since two pores are formed in the cell, the extracellular solution easily passes through the cell, so that the solution is efficiently taken into the cell. Since the gene is dispersed in an extracellular solution, a sufficient amount is ensured in the cell for expression in the cell, and high introduction efficiency is obtained.

  (2) The gene introduction device according to (1), wherein the perforation means is a needle part having a fine needle inserted into the cell and a support part for supporting the fine needle.

(Corresponding embodiment)
The first and second embodiments correspond to the embodiment related to the gene introduction device described in (2). In these embodiments, the introduction needle 44 or 441, 442 corresponds to the fine needle, the silicon base portion 58 corresponds to the support portion, and the cantilever 52 corresponds to the needle portion.

(Function and effect)
According to the gene introduction device described in (2), since a fine needle is used, damage to cells can be suppressed, and genes can be introduced into target cells.

  (3) The gene introduction device according to (2), wherein the fine needle penetrates the cell and perforates the cell membrane.

(Corresponding embodiment)
The embodiment related to the gene introduction device described in (3) corresponds to the first embodiment.

(Function and effect)
According to the gene introduction device described in (3), since the pore is formed through the nucleus of the cell, the gene circulates in the nucleus and is efficiently taken into the nucleus.

  (4) The gene according to (3), wherein the driving unit moves the fine needle further in the direction of the substrate than the position where the tip of the fine needle is in contact with the substrate holding the cell. Introduction device.

(Corresponding embodiment)
The embodiment relating to the gene introduction device described in (4) corresponds to the first embodiment. In the embodiment, the substrate 68 of the workpiece 14 corresponds to the substrate.

(Function and effect)
According to the gene introduction device described in (4), the fine needle can be slid with respect to the substrate surface by lowering the fine needle further than the contact position with the substrate. The two places on the upper surface side and the bottom surface side can be reliably broken.

  (5) The driving unit moves the fine needle in a direction parallel to the substrate at a position where the tip of the fine needle is in contact with the substrate holding the cell. Gene transfer device.

(Corresponding embodiment)
The embodiment relating to the gene introduction device described in (5) corresponds to the first embodiment.

(Function and effect)
According to the gene introduction device described in (5), the fine needle can be slid with respect to the substrate surface by horizontally moving the fine needle in contact with the substrate. Two places on the upper surface side and the bottom surface side can be reliably broken.

  (6) The gene introduction device according to (2), wherein the fine needle perforates the cell membrane from at least two places from the outside of the cell.

(Corresponding embodiment)
The embodiment related to the gene introduction device described in (6) corresponds to the second embodiment.

(Function and effect)
According to the gene introduction device described in (6), it is possible to reliably introduce a gene even in a cell having a weak adhesive force with a substrate. In addition, even in a state where cells are stacked, it is possible to select only the cells on the upper side and introduce a gene.

  (7) The gene introduction device according to (6), wherein the fine needle perforates the cell membrane at least twice.

(Corresponding embodiment)
The embodiment relating to the gene introduction device described in (7) corresponds to the second embodiment.

(Function and effect)
According to the gene introduction device described in (7), at least two holes can be drilled with one fine needle.

(8) The punching means has at least two needle portions,
The gene introduction apparatus according to (6), wherein each of the fine needles of the needle portion perforates the cell membrane of the same cell.

(Corresponding embodiment)
The embodiment relating to the gene introduction device described in (8) corresponds to the second embodiment.

(Function and effect)
According to the gene introduction device described in (8), at least two holes can be drilled in one operation.

  (9) The gene introduction device according to (1), wherein the perforating means is a laser that perforates the cell membrane by penetrating laser light through the cell and adjusting a focal position to two locations of the cell membrane. .

(Corresponding embodiment)
The embodiment relating to the gene introduction device described in (9) corresponds to the third embodiment. In that embodiment, a laser light source 96 corresponds to the laser.

(Function and effect)
According to the gene introduction device described in (9), since the perforation is performed by the laser beam, the perforation penetrating the cell can be formed with minimal invasiveness. Further, since maintenance such as replacement of a fine needle that contacts a cell and sterilization is unnecessary, it is easy to maintain performance.

(10) selecting a cell into which a gene is to be introduced;
Perforating at least two portions of the cell membrane of the cell;
A gene transfer method characterized by comprising:

(Corresponding embodiment)
The first to third embodiments correspond to the gene transfer method described in (10).

(Function and effect)
According to the gene introduction method described in (10), since two pores are formed in the cell, the extracellular solution easily passes through the cell, so that the solution is efficiently taken into the cell. Since the gene is dispersed in an extracellular solution, a sufficient amount is ensured in the cell for expression in the cell, and high introduction efficiency is obtained.

FIG. 1 is an overall configuration diagram showing a gene introduction apparatus according to the first embodiment of the present invention. FIG. 2A is an enlarged view showing the structure near the cantilever, and FIG. 2B is an enlarged view showing the structure of the cantilever. FIGS. 3A to 3C are diagrams showing a series of states of the introduction needle provided on the cantilever for explaining the gene introduction method according to the first embodiment of the present invention. FIG. 4 (A) is a view showing a microscope image obtained by immersing HelaS3 cells in a fluorescent dye solution and observing with a laser scanning microscope, and FIG. 4 (B) shows the gene introduction apparatus according to the first embodiment and It is a figure which shows the microscope image of the result of having introduce | transduced the dye solution in the cell by the method. FIG. 5A is a view showing a microscopic image immediately after introducing a gene expressing a GFP fluorescent protein into Hela S3 cells by the gene introduction apparatus and method according to the first embodiment, and FIG. 5B and FIG. ) Are diagrams showing a phase difference observation microscope image after 24 hours and a microscope image by fluorescence observation, respectively. FIG. 6 is an enlarged view showing the structure of the cantilever in the gene introduction device according to the second embodiment of the present invention. FIG. 7 is a diagram showing a state of insertion of an introduction needle provided on a cantilever into a cell for explaining a gene introduction method according to the second embodiment of the present invention. FIG. 8 is a diagram showing two perforations formed in the cell membrane on the upper surface of the cell. FIG. 9 is a configuration diagram showing a main part of the gene introduction device according to the third embodiment of the present invention. FIG. 10 is a diagram illustrating a display example of cells and spot positions. FIG. 11 is a diagram showing a flowchart showing the procedure of the gene introduction method using the gene introduction apparatus according to the third embodiment of the present invention.

Explanation of symbols

    DESCRIPTION OF SYMBOLS 10 ... Gene transfer apparatus, 12 ... Inverted microscope, 14 ... Workpiece, 16 ... Work holder, 18 ... Illumination device, 20 ... Observation apparatus, 22 ... Microscope XY stage controller, 24 ... Microscope XY stage controller, 26 ... Microscope controller, 28 DESCRIPTION OF SYMBOLS ... Image processing apparatus 30 ... Monitor 32 ... Control computer 34 ... Transmission illumination light source 36 ... Condenser lens 38 ... Epi-illumination light source 40 ... CCD camera 42 ... Eyepiece lens 44,441,442 ... Introduction needle, 46 ... Introduction needle holder, 46A ... Arm, 48 ... Drive unit, 50 ... Control box, 52 ... Cantilever, 54 ... Mounting member, 56 ... Z-axis actuator, 58 ... Silicon base part, 60, 601, 602 ... Lever part 62 ... Drive unit control circuit, 64 ... Ack Control circuit, 66 ... DNA dispersion solution, 68 ... substrate, 70 ... cell, 72 ... cell membrane, 74 ... nucleus, 76 ... break point, 78 ... perforation, 80 ... mount, 82 ... laser pointer, 84,86 ... half mirror 88, 98 ... mirror, 90 ... objective lens, 92 ... actuator, 94 ... spot position, 96 ... laser light source.

Claims (10)

In a gene introduction apparatus for introducing a gene into a cell,
A perforation means for perforating a cell membrane of a cell cultured in a culture solution in which the gene to be introduced is dispersed;
Driving means for driving the perforation means to perforate at least two locations of the cell membrane;
A gene introduction device comprising:   2. The gene introduction device according to claim 1, wherein the perforating means is a needle part having a fine needle inserted into the cell and a support part for supporting the fine needle.   The gene introduction device according to claim 2, wherein the fine needle penetrates the cell and perforates the cell membrane.   4. The gene transfer device according to claim 3, wherein the driving unit further moves the fine needle in a direction toward the substrate rather than a position where a tip of the fine needle is in contact with a substrate holding the cells.   The gene introduction device according to claim 3, wherein the driving unit moves the fine needle in a horizontal direction with respect to the substrate at a position where a tip of the fine needle is in contact with the substrate holding the cell. .   The gene introduction device according to claim 2, wherein the fine needle perforates the cell membrane from at least two places from the outside of the cell.   The gene introduction apparatus according to claim 6, wherein the fine needle perforates the cell membrane at least twice. The perforating means has at least two of the needle portions,
The gene introduction apparatus according to claim 6, wherein the fine needles of the needle part each pierce the cell membrane of the same cell.   2. The gene introduction apparatus according to claim 1, wherein the perforating means is a laser that perforates the cell membrane by penetrating laser light through the cell and adjusting a focal position to two locations of the cell membrane. Selecting a cell into which the gene is to be introduced;
Perforating at least two portions of the cell membrane of the cell;
A gene transfer method characterized by comprising:

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