JPH05192171A - Microinjection method and its apparatus - Google Patents

Abstract

(57) [Summary] [Purpose] To accurately inject a very small amount of a sample such as DNA into a cell. An electrophoretic unit 2 for injecting a sample into cells by electrophoresis, a power supply unit 1 for applying a high voltage to the electrophoretic unit 2, and a coarse moving unit 1 for driving a distal end of the electrophoretic unit 2 having a large moving distance.
0 and a fine movement unit 11 for a micro area, a controller unit 4 that controls the operation of the drive unit 3 in the xyz directions, a microscope unit 5 having a television camera, and a microscope image enlarged by the microscope unit 5 is projected. It is composed of a television monitor section 6 and a computer section 7 for inputting information of the television monitor section 6, moving the tip of the electrophoretic section 2 to a designated position of the television monitor section 6, and outputting an injection command to the controller section 4. ing. [Effect] By using the electrophoresis method for injecting the sample into the cells, it is possible to precisely inject a very small amount of sample by controlling the applied voltage and the voltage application time.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a microinjection method for directly injecting a sample such as a gene or gene product into a cell by using an electrophoresis method.

[0002]

2. Description of the Related Art When it is desired to inject a sample such as DNA into cells, a microinjection method of mechanically injecting air or a high voltage of several thousand V / cm to a cell suspension for several tens of microseconds is used. Utilizing the fact that external fluid is taken in through small pores that occur in the cell membrane for a short time when pulsed, a sample to be injected such as DNA is added to the extracellular fluid, and electroporation is used to introduce this into cells. Was there.
The microinjection method is described, for example, in Methods in Enzymology, Volume 65 (1
980, pp. 816-825 (Methods)
in Enzymology, Vol. 65 (198
0) pp. 816-825), regarding electroporation, The Journal of Biological Chemistry, 264, No. 26 (1989), pages 15494 to 15500 (The Journal).
al of Biological Chemistr
y, Vol. 264, No. 264. 26 (1989) pp. 1
5494-15500).

[0003]

In the conventional microinjection method of mechanically injecting a sample into cells by air pressure, since the volume of the sample to be introduced is adjusted by air pressure, it is extremely high on the order of picoliters. It was difficult to introduce a small amount of sample. Furthermore, the tip of the microinjector is depressurized to suck extracellular fluid or air into the injector, which causes a problem that the amount of the sample injected into the cell is not constant. In the electroporation method, the concentration of the sample to be injected is determined by the concentration of extracellular fluid, but the volume is quantitative because it is difficult to control the diameter and life of the pores formed when an electric pulse is applied. It was difficult to inject the sample into cells. Furthermore, in both cases, the microinjection method and the electroporation method have a problem that the intracellular environment such as the intracellular volume and the ratio of constituents is changed because the sample to be injected is injected into the cell as an aqueous solution. It was

[0004]

[Means for Solving the Problems] In order to accurately inject a very small amount of sample into cells, the force for injection is based on the electrophoresis method. In addition, it is necessary to prevent suction of cell liquid and air due to depressurization of the tip of the glass capillary injector, and to suppress unnecessary liquid flow caused by electrophoresis, that is, inflow of liquid other than the sample into cells due to electroosmotic flow. Accordingly, glass having pinholes was used as the partition wall. In addition, in order to inject a sample into a specific area of a cell having a size of about 10 μm with high positional accuracy, φ1.0 μ
A glass capillary injector of about m was used.

[0005]

The principle of the electrophoresis method is shown in FIG. The electrophoresis method uses a capillary 21 that performs electrophoresis using the power supply unit 1.
Charged substance 2 by applying high voltage across both ends of
It is one of the analytical methods that separates according to the difference in mobility.
In the electrophoretic method, the movement of substances depends on the applied voltage and time. Therefore, by controlling the applied voltage and the voltage application time, it is possible to control the amount of movement of even a very small amount of substance. Therefore, even a very small amount of sample can be accurately injected into a minute area such as a cell. Furthermore, by using an injector with a diameter of about 1.0 μm, it is possible to accurately inject a sample into a specific portion of a cell with a size of about 10 μm. In general,
In electrophoresis using a glass capillary, a liquid flow occurs in the capillary due to electroosmotic flow. An explanation of electroosmotic flow is shown in FIG. An electric double layer 24 is formed by the silanol group 23 on the surface of the glass capillary 21,
An electroosmotic flow 25 is generated to generate a liquid flow. Therefore, in addition to the transfer of the substance by the original electrophoresis method, the transfer of the substance by the electroosmotic flow occurs. High injection accuracy can be obtained by the electrophoresis injection method, but if a higher injection accuracy is required, or if a very small amount of solution needs to flow into cells, A conductive partition may be provided. A description of the conductive partition between the electrodes is shown in FIG. A glass plate 27 having several tens of Å pinholes 26 is installed between the capillaries 21 for electrophoresis. When a voltage is applied to both ends of the capillary 21 by the power supply unit 1, the low molecular weight ions 28 move. At this time, the low-molecular weight ions 28 can pass through the pinhole 26, so that an electric current flows and electrophoresis is performed. However, migration of the solution due to electroosmotic flow is impeded by the loading of pinhole 26. Therefore, by using glass having pinholes, the conductivity is maintained and electrophoresis is performed, but the inflow of the solution into the cells due to the electroosmotic flow can be suppressed. Furthermore, since one end of the glass capillary is closed by the conductive partition, the decompression at the tip of the glass capillary is also suppressed, and the suction of extracellular fluid and air into the glass capillary is reduced, significantly improving the accuracy of the injected sample volume. can do. The partition wall is not limited to the glass plate 27, and for example, a partition wall having a minute communication hole known as a sintered filter can be used. In essence, it is sufficient if it is an obstacle to the movement of the fluid and is electrically conductive.

[0006]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS An embodiment of the present invention will be described below with reference to the block diagram of FIG.

The microinjector includes information about the power source unit 1, the electrophoretic unit 2, the driving unit 3, the controller unit 4 for controlling the driving unit 3, the microscope unit 5, the television monitor unit 6, and the television monitor unit 6 in the controller unit 4. Computer section 7 to output to
It consists of The power supply unit 1 has an output voltage of 0-30k
At V, using a high-voltage power supply with easy polarity switching, using electrodes 41 and 42 between the sample tank 8 and the buffer tank 9, and a sample container 17 and a buffer tank arranged in parallel in the sample tank 8. A voltage is applied between the electrodes 9 and 9 by using the electrodes 41 and 43. The polarity is determined to be reversed depending on which of the voltages is applied. Electrophoresis unit 2 is φ1.0m
A fused silica glass tube of m was stretched by a stretching device (for example, a microelectrode maker, Narimo PN-3) by a stretching force by heat and an electromagnetic field, and a glass capillary having a tip of φ1.0 μm was used. When it is desired to suppress the inflow of the solution into the cells due to high injection accuracy or electroosmotic flow, a conductive partition may be provided in the middle of the electrophoresis section 2, that is, between the sample tank 8 and the buffer tank 9. The driving unit 3 is composed of a coarse moving unit 10 having a long moving distance for driving a pulse motor and a fine moving unit 11 for a minute area driven by a piezo element having a driving resolution of several 10Å, and controls the movement in the xyz directions. The fine movement unit 11 may use a hydraulic micromanipulator when the minimum drive distance is about 1.0 μm. The controller unit 4 controls the operation in the xyz directions by the drive unit 3, and general-purpose operations such as sample suction can be performed with a single switch by programming the moving distance in the xyz direction and the stop time. it can. In the microscope section 5, the light from the light source 12 for illumination illuminates the sample tank 8 via the condenser lens 13, and the sample in the sample tank is reflected by the reflection mirror 14.
The image is taken by the television camera 16 via 15. The operation of injecting the sample into the cells is performed by designating the cells displayed on the television monitor unit 6 with a light pen or the like, and the designated position is input to the computer unit 7 as position information.
The computer section 7 outputs movement information based on the position information to the controller section 4, and the controller section 4 controls the drive section 3 based on the position information. Therefore, the sample is automatically injected by designating the cells displayed on the television monitor unit 6 with a light pen or the like. The operator gives position information to the controller unit 4 via the computer unit 7 while looking at the finder 44 of the television monitor unit 6 or the microscope unit 5, and outputs movement information based on the position information to output the controller unit. By controlling
It is also possible to manually inject the sample.

The sample is injected into the cells by first moving the tip of the glass capillary of the electrophoretic unit 2 to the sample container 17 and applying a voltage of a predetermined polarity to the electrodes 41 and 43 by the power supply unit 1. A fixed amount of the sample in the sample container 17 installed in the microscope unit 5 was aspirated from the tip of the glass capillary of the electrophoresis unit 2 by electrophoresis. The amount of sample to be sucked can be easily determined on the picogram order by the applied voltage and the voltage application time. next,
The tip of the glass capillary of the electrophoretic unit 2 is moved to the sample tank 8. This is done by designating a specific portion of the cells displayed on the television monitor unit 6 with a light pen, so that the computer unit 7 causes the controller unit 4 to move. The position information of the navel was given to automatically move the tip of the electrophoretic section 2 to a specific portion of the cell. Then, the polarity of the power supply unit 1 was switched, a predetermined voltage was applied to the electrodes 41 and 42, and the sample was introduced into the cells by electrophoresis. The sample injection amount can be determined by the applied voltage and the voltage application time. When performing the above operation manually, a signal is given from the computer section 7 to the controller section 4 while looking at the monitor screen 6 or the finder 44, and the tip of the electrophoretic section 2 is roughly moved by using the coarse movement section 10. Positioning quickly,
Then, the fine movement part 11 was used to position the cell on a specific part. After that, the polarity of the power supply unit 1 is switched and the electrode 4
A predetermined voltage was applied to 1 and 42, and the sample was introduced into the cells by electrophoresis.

Next, a case where the electroosmotic flow is removed as necessary in the embodiment of the present invention will be described with reference to FIG.

As described above, in order to remove the electroosmotic flow, the electrophoretic section 2 is provided with a conductive partition that resists the movement of the fluid and is electrically conductive. Other configurations may be the same as in FIG. As the conductive partition 32, a porous glass plate (made by Corning) having pinholes was used. The glass capillary 31 is arranged so as to sandwich the conductive partition 32, and this portion is a Teflon seal 3 for preventing leakage.
Closely attached by 3. Teflon seal 33 is 2 up and down
It is divided and each is held by the holder 34 which is also divided into two. The cylinder 35, the piston 36, the spring 37, and the outer box 50 act on the two holders 34 to push them up and down. And a pressurizer 35.

When there is no problem even if there is an electroosmotic flow, or when it is desired to utilize this partition, the lower holder is pushed down to remove the conductive partition 32, and then the glass capillary 31 is attached to the Teflon seal 33. It is sufficient that the conductive partition 32 is removed from the configuration shown in FIG. The two glass capillaries 31 are in close contact with each other by the Teflon seal 33, and there is no leakage.

[0012]

According to the present embodiment, since the sample is injected into a minute area such as a cell by using the electrophoresis method, it is possible to accurately inject even a very small amount by controlling the applied voltage and the voltage application time. There is an effect that can be.

[Brief description of drawings]

FIG. 1 is a block diagram showing a configuration of a microinjection device according to an embodiment of the present invention.

FIG. 2 is a cross-sectional view showing the structure of a cassette-type conductive partition that can be used in an embodiment of the present invention.

FIG. 3 is an explanatory view of the principle of the electrophoresis method.

FIG. 4 is an explanatory diagram of the principle of electroosmotic flow.

FIG. 5 is an explanatory diagram of a conductive partition.

[Explanation of symbols]

1 ... Power supply part, 2 ... Electrophoresis part, 3 ... Driving part, 4 ... Controller part, 5 ... Microscope part, 6 ... Television monitor part, 7 ... Computer part, 8 ... Sample tank, 9 ... Buffer tank, 10 ... Coarse Moving part, 11 ... Fine moving part, 12 ... Illuminating light source, 13 ... Condenser lens, 14, 15 ... Reflecting mirror, 16 ... Television camera, 17 ... Sample container.

Claims (6)

[Claims] 1. A microinjection method, which comprises injecting a sample into a microscopic region such as a cell using an electrophoresis method. 2. A microinjection device comprising: a power supply unit for applying a voltage, a migration unit for electrophoresis of a sample, and a drive unit for moving one end of the migration unit in correspondence with the position of the injection target. 3. A power supply unit for applying a voltage, an electrophoretic unit for electrophoresing a sample, a drive unit for moving one end of the electrophoretic unit corresponding to the position of the injection target, and an area including the injection target of the sample is enlarged. A microscope, a television monitor that captures an enlarged image of an area including the injection target of the sample by the microscope, a position designating device that designates a specific position on the television monitor, and position information designated by the position designating device. A microinjection device comprising: a means for giving to the drive part, and a means for performing sequence control so as to apply a force by electrophoresis after moving one end of the electrophoretic part with respect to an injection target. 4. The microinjection device according to claim 3, wherein the electrophoretic section is provided with a conductive partition wall. 5. The microinjection device according to claim 4, wherein the conductive partition wall is glass having pinholes. 6. The conductive partition wall according to claim 5,
A microinjection device, which is detachably provided.