JP2002065241A - Apparatus for introducing sample intoamphibian oocyte - Google Patents

Abstract

PROBLEM TO BE SOLVED: To introduce a sample into amphibian oocytes at a prescribed depth with a good accuracy and retain the quality of the oocytes or a position where a needle is inserted as information. SOLUTION: This apparatus for introducing a sample into amphibian oocytes is characterized as comprising a tray for holding plural amphibian oocytes, the introduction needle for introducing the sample into the plural amphibian oocytes, a driving part for moving the relative position of the tray and the introduction needle and a controlling part for inputting the depth of the introduction needle relatively to the tray or the amphibian oocytes during the introduction of the sample and introducing the sample into the plural amphibian oocytes at the depth.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an automatic apparatus for introducing a sample of a gene, a pigment, a protein, a peptide, a drug, etc. into an amphibian oocyte such as a frog using a pipette-like needle. A method for introducing a sample of a gene, a pigment, a protein, a peptide, a drug, etc. into a specific position of an amphibian oocyte, introducing a sample into an amphibian oocyte of which quality is guaranteed with respect to the sample introduction, and a specific position and depth. And a method of selling or transferring the amphibian oocyte thus obtained.

[0002]

2. Description of the Related Art Frog oocytes, such as Xenopus, into which samples of genes, pigments, proteins, peptides, drugs, etc. have been introduced, are relatively large and can be obtained in large quantities at low cost. Pigments, proteins, peptides,
It is widely used for purposes such as confirming the action of drugs, analyzing gene functions, and producing proteins as gene products.However, conventionally, individual researchers breed frogs and collect oocytes. I was going.

Conventionally, genes, pigments, proteins, peptides,
When introducing a sample of a drug or the like into an oocyte of an amphibian such as a frog, a technician manually inserts the pipette filled with the sample into the oocyte under a microscope using a manipulator. Was. The pipette is attached to the injector,
A fixed amount of sample is discharged into cells by hydraulic pressure or air pressure. As a method of applying a voltage and discharging a fixed amount of sample into cells, there are JP-A-5-192171 and JP-A-6-343478. Each method is a technique in which a needle is manually brought close to a cell while a cell is observed under a microscope to introduce a sample.

[0004]

The manual introduction of genes and the like described above depends on the skill and skill of the individual engineer.
There was a problem that the percentage of oocytes into which the sample could be introduced fluctuated. This is one of the reasons that the number of oocytes that can be introduced into a sample per fixed time varies from engineer to engineer, and the degree of metamorphosis of the sample varies from engineer to engineer. An object of the present invention is to make the number of processes per hour constant regardless of the skill and skill of a technician.

[0005] Further, in the above-mentioned prior art, no consideration is given to unifying the sample introduction depth into the oocyte, and it is difficult even for a skilled technician to control the depth. As a result, sample introduction into specific intracellular organelles such as the nucleus has to rely on chance. An object of the present invention is to facilitate introduction of a sample into an organelle capable of being controlled in the depth direction into a nucleus or the like by unifying the sample introduction depth.

[0006] Furthermore, in the above-mentioned prior art, no consideration is given to preserving cell information at the time of introducing a sample, and it is difficult to obtain a correlation between the sample introduction information and the subsequent cell reaction. Was. It is therefore an object of the present invention to obtain these correlations.

[0007] In addition, conventionally, since oocytes were individually procured, it was impossible to mass-produce them or produce them at the right timing according to demand. Therefore, another object of the present invention is to produce, sell and transport oocytes into which a specific sample has been introduced, or oocytes which have been guaranteed to have a specific sample introduced into a certain position.

[0008]

SUMMARY OF THE INVENTION In order to solve the above-mentioned problems, the present invention uses a pipette-like needle to convert a sample of genes, dyes, proteins, peptides, drugs, etc. into an amphibian oocyte such as a frog. It is an object to provide a device for introducing an arbitrary position and an arbitrary depth by automatic control.

That is, the present invention provides a tray for holding a plurality of amphibian oocytes, an introduction needle for introducing a sample into the plurality of amphibian oocytes, and a drive unit for moving a relative position between the tray and the introduction needle. And a control unit for inputting the depth of the introduction needle with respect to the tray or the amphibian oocyte during sample introduction and controlling the movement, and introducing a sample into the plurality of amphibian oocytes at the depth. The present invention provides an amphibian oocyte sample introduction device characterized in that:

[0010] Further, the present invention provides a tray for holding a plurality of amphibian oocytes, an introduction needle for introducing a sample into the plurality of amphibian oocytes, and a relative position between the tray and the introduction needle in a three-dimensional direction. A drive unit for moving the control unit, a control unit for controlling the movement, an information acquisition unit for obtaining visual information of the amphibian oocyte at the time of introduction, and a storage unit for accumulating the information, Provided is an amphibian oocyte sample introduction system, which is introduced into a plurality of amphibian oocytes. As a result, it has become possible to rapidly introduce a sample into a plurality of amphibian oocytes at a substantially constant depth.

Further, the tray has a hole characterized by a cylinder having a flat bottom surface or a conical bottom portion and having a maximum diameter of 105 to 150% of the diameter of the plurality of amphibian oocytes. As a result, about 80% of a plurality of oocytes on a tray can be introduced into the same plane without using any other special means.

[0012] The present inventors have found that when mRNA is introduced into an oocyte, the mRNA is introduced into the black surface of the oocyte and the mRNA is introduced into the white surface.
When NA was introduced, it was found that the function expression efficiency of the protein was different. That is, in order to suppress the variation in the efficiency of protein function expression between oocytes, it is important to collect oocytes transfected with mRNA on the same surface. According to the present invention, it is possible to save information of cells at the time of introducing a sample and easily derive a correlation with a subsequent cell reaction.

Further, the present invention provides an apparatus having a tray for holding a plurality of amphibian oocytes and an introduction needle for introducing a sample into the plurality of amphibian oocytes, wherein the tray or the amphibian oocyte is used. Setting the depth of the introduction needle to a first depth with respect to, the first oocyte of the plurality of amphibian oocytes, the sample with the introduction needle to the first oocyte
The step of automatically moving the relative position between the tray and the introduction needle, and then the introduction needle to the second oocyte of the plurality of amphibian oocytes Introducing a sample to the first depth. An automatic amphibian oocyte sample introduction method is provided.

Further, the present invention provides an apparatus having a tray for holding a plurality of amphibian oocytes and an introduction needle for introducing a sample into the plurality of amphibian oocytes. A step of introducing a sample into the first oocyte with the introduction needle, a step of moving the relative position of the tray and the introduction needle, and then a second of the plurality of amphibian oocytes A step of introducing a sample into the oocyte with the introduction needle, a step of obtaining the state of the oocyte at the time of sample introduction as visual information, and having a step of accumulating the visual information, An automatic amphibian oocyte sample introduction method is provided.

According to the invention of the amphibian oocyte sample introduction apparatus or the introduction method, the present invention further provides a plurality of amphibians characterized in that the respective samples are introduced under the condition that the sample introduction depths are substantially equal. Provide an oocyte.
Further, the present invention provides the above amphibian oocyte, wherein the sample is introduced under substantially the same conditions as the introduction position.

Further, the present invention provides the following.
While using a device having a tray holding a plurality of amphibian oocytes and an introduction needle for introducing a sample into the plurality of amphibian oocytes, while moving the relative position of the introduction needle with respect to the tray, the A sample is introduced into each of the amphibian oocytes with the introduction needle, the visual information of each of the plurality of amphibian oocytes at the time of the introduction is obtained, and the plurality of amphibian oocytes are obtained based on the visual information. The amphibian egg into which the sample has been introduced, wherein the introduction is performed by collecting a plurality of oocytes introduced on the black side of the oocyte or a plurality of oocytes introduced on the white side of the oocyte. A method for producing a mother cell group.

[0017] A plurality of amphibian oocytes into which the sample has been introduced under the condition that the sample introduction depths are substantially equal are set, and information on the sample introduction into the plurality of amphibian oocytes is attached. Selling or transferring a plurality of amphibian oocytes.

Alternatively, a plurality of amphibian oocytes, each of which has been introduced under the condition that the sample introduction depths are substantially equal, are placed in a container, and information on sample introduction into the plurality of amphibian oocytes is written in the container. A method of selling or transferring a plurality of amphibian oocytes with a label attached.

Here, the information on sample introduction into the plurality of amphibian oocytes includes at least the date and time of introduction, the quality assurance period of the oocyte, the position at which the sample was introduced, the depth at which the sample was introduced, and the probability of expression. It concerns one of them. Thus, according to the present invention, it has become possible to obtain a plurality of oocyte groups having substantially the same conditions for introduction together with their information.

[0020]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be further described below with reference to the drawings. FIG. 1 shows the principle of this device. The tray for aligning the oocytes is, for example, 12
And a total of 96 trays with holes having a uniform depth and shape in the orthogonal direction can be used, but the number of holes in the tray is not necessarily limited to 96. Amphibian oocytes are generally heavier on the white side than on the black side. Therefore, by making the hole diameter of the tray slightly larger than the oocyte using the tray, the oocytes aligned on the tray do not change direction due to rotation or the like, and about 80% of the cells are held on the black side on average Thus, the probability of introduction on the same plane can be increased without using any other special means. The shape of the hole for aligning the oocytes is preferably a cylindrical shape having a circular bottom surface and a constant cross-sectional shape parallel to the bottom surface from the bottom surface to the opening, or a conical bottom shape. The diameter of the opening of the hole should be equal to or greater than the diameter of the amphibian oocyte used. Further, it is preferable that there is room for the oocyte to rotate in the above hole filled with physiological saline or the like for the reason that the oocyte is well. Specifically, 105 to 150% of the diameter of the oocyte to be used is set to the maximum of the hole. It has been experimentally confirmed that the diameter is desirable. For example, Xenopus oocytes have a diameter of about 1.
Since the diameter is about 3 mm, by setting the diameter of the hole to about 1.4 to 2 mm, cells can be fixed in a specific direction without damaging the cells. The hole shape suitable in the present invention is, for example, as shown in FIG.
It is a mortar type with a depth of 1.4 mm and a depth of 0.56 mm in the cylindrical part. In addition to using the tray, a dropper or the like may be used to further increase the probability of introduction on the same surface, or to operate such that a specific surface, for example, a white surface is on the upper side.

Examples of the sample to be introduced include, but are not particularly limited to, genes, dyes, proteins, peptides, drugs and the like. Further, in the following examples, an oocyte into which human histamine receptor cRNA has been introduced is described, but the gene used for introduction is not limited to cRNA.
And an RNA or a synthesized oligonucleotide. The needle for introducing the sample is preferably a pipette-like needle, but is not particularly limited. Also,
The digital camera 8 is used to acquire cell information such as the orientation of the oocyte, and the means for detecting that the introduction needle 6 has contacted the oocyte surface is based on visual information through the CCD camera 7 as an example. However, the means necessary to obtain such information is not limited to the digital camera 8 and the CCD camera 7. For example, by installing a sensor that detects changes in pressure, temperature, electricity, humidity, pH, etc. into the introduction device,
The cell surface can be detected based on such information.

The oocytes before the sample introduction are arranged in the holes of the tray 9, and after filling the tray 9 with a physiological saline solution 14 for amphibians, the oocyte is placed on the orthogonal moving table 11 and the horizontal moving table 12. It is preferable that the position of the oocyte 13 into which the sample is introduced from the introduction needle 6 is determined by controlling the movement of the orthogonal carriage 11 and the horizontal carriage 12 in the X-axis direction and the Y-axis direction by the control device 1. However, contrary to the configuration of FIG. 1, the tray 9 is fixed and the introduction needle 6
Can be configured to be movable.

When the tray 9 is at the position indicated by the broken line, the oocyte is photographed by the digital camera 8 and the photographing data is sent to the control device 1 to accumulate information such as the quality and orientation of the oocyte. Can be.

The horizontal moving table 12 and the orthogonal moving table 11 are connected to the control unit 1
To move the center of the first oocyte 13 at a predetermined position in the oocytes arranged on the tray 9 to a position below the gene transfer needle 6. Here, the introduction needle moving table 4 operates in accordance with the instruction of the control device 1 to move the introduction needle in the Z-axis direction, and the tip of the introduction needle 6 attached to the introduction device 5 is slightly separated from the surface of the oocyte. To a position, for example, a few hundred mm before. Here, while observing the image taken by the CCD camera 7 on the monitor 3, a command is issued by the control auxiliary device 2,
The needle moving table 4 is lowered at a low speed. Visual information, pressure change, temperature change, electrical change, humidity change, pH change, etc., detects that the tip of the introduction needle 6 has contacted the surface of the oocyte 13,
At this position, the introduction needle moving base 4 is stopped. This position serves as a reference point for the subsequent gene transfer operation. Control position 1
And perform the following operations. That is, the moving distance and depth of the introduction needle in the direction perpendicular to the surface on which the tray is placed from the reference point to the position for introducing the sample are set, and the introduction needle 6 is inserted at the set depth. To discharge a fixed amount of the sample. For sample introduction, for example, Z-axis control such as lowering the introduction needle by 0.2 mm below the position where the introduction needle 6 is in contact with the surface of the oocyte 13 can be performed. The optimal introduction depth of the introduction needle 6 into the cell differs depending on the type of the sample to be introduced and the purpose of the introduction, and can be set as appropriate. If the introduction depth is too shallow, the sample will not spread into the cells, and if it is too deep, the probability of damaging the nucleus or damaging the cells will increase. Therefore, it is desirable to introduce a sample at a substantially constant depth from the viewpoint of expression efficiency. For example, if you want to introduce mRNA into the cytoplasm and express the protein,
It is desirable to insert a needle at a depth of 0.02 to 0.1 mm from the cell surface. On the other hand, when it is desired to introduce DNA into the nucleus and express the protein, it is desirable to insert a needle at a depth of 0.05 to 0.2 mm from the cell surface. However, since the shape of the oocyte is slightly distorted by the contact of the needle at the time of introduction, the sample is actually introduced at a position shallower than the set depth. The time for introducing the sample is controlled by setting the time during which the needle is injected into the cell according to the amount of injection into the cell and the like. In order to further improve the introduction efficiency, a plurality of introduction needles 6 may be provided. In this case, it may be sufficient for the drive unit of the apparatus to move the relative position between the introduction needle and the tray in one-dimensional or two-dimensional directions.

Thereafter, based on the three-dimensional position information of the first oocyte, the specified time, speed, and speed are assigned to an arbitrary number of oocytes after the second in the tray 9 by automatic control. Automatically introduce the sample at the introduction depth. In addition, since the size of the oocyte may vary, a function of detecting the surface position each time the oocyte is introduced may be provided. In addition, the cell information of the oocyte can be stored in a computer, and can be extracted when necessary.

Furthermore, the movement of the introduction needle 6 and the oocyte 13 at the time of sample introduction, visual information of the oocyte at the time of introduction, and the like are stored in a computer.
It can also be set to read the depth, cell characteristics, etc. In this case, it is desirable that the visual information on each oocyte is associated with the position on the tray by assigning a number or the like.

Also, it has been known that amphibian oocytes have a white surface and a black surface, and it has been known that their functions are different from each other.
It was found that when NA was introduced, the efficiency of protein function expression was different between the case where mRNA was introduced into the black surface of the oocyte and the case where mRNA was introduced into the white surface. That is, when histamine receptor mRNA is introduced, oocyte cells having a higher expression efficiency and better ligand responsiveness can be obtained by introducing histamine receptor mRNA into a black surface than when introducing histamine receptor mRNA into a white surface. On the other hand, when a protein containing a chromophore, a fluorescent protein, or a gene or a dye thereof is introduced, color and light information can be obtained with higher sensitivity by introducing the protein on a white surface. By using the tray as described above, about 80% of the cells on the tray can be introduced into the black surface. Alternatively, when it is desired to obtain cells in which a sample has been introduced only on a specific surface of a cell, for example, only on a black surface or only on a white surface, as described above, the individual The cells are manipulated using a dropper or the like. Alternatively, at the time of sample introduction, visual information detecting means or a black-and-white discriminating sensor was used to acquire the sample introduction position information on the cell surface, and from the acquired information, the sample was introduced only into cells whose target surface was on the upper side. Cells can also be collected. As a result, an oocyte group having substantially the same conditions for the introduction position is obtained. In the present invention, the “specific position” refers to, for example, a position on the black side, the white side, or near the equator of the oocyte.

By using the device having the above configuration,
A sample can be introduced into a specific position and depth of an amphibian oocyte, and the function expression efficiency of the introduced sample (introduction efficiency)
Can produce oocytes of almost the same quality quickly and in large quantities. Accordingly, the present invention also provides a method for introducing a sample to a specific location and depth of an amphibian oocyte using the device of the present invention.

It has been found that the use of the apparatus of the present invention improves the sample introduction efficiency as follows. In other words, for a beginner who has no experience of manually introducing a sample, it takes about 30 minutes to introduce the sample into 25 cells, and the introduction efficiency is about the expression rate when the gene is used as the sample.
Although 30%, the use of the device of the present invention requires only 3 minutes to introduce a sample into 25 cells, and the introduction efficiency reaches about 80%. In the case of a skilled person who introduces a sample by manual operation, the time required for the introduction is not so shortened, but the introduction efficiency is, for example, 80% manually.
In the case of%, it can be further increased to 90% by use of the apparatus.

Thus, by using the apparatus and method of the present invention, about 80 to 80, independent of the skill of the operator.
A transfer efficiency of 90% was achieved, and the present invention made it possible for the first time to sell or transfer a plurality of oocytes with controlled sample transfer conditions. Thus, in another aspect, the present invention provides an amphibian oocyte in which the introduction of a sample to a specific location and depth is guaranteed.

In addition, it is possible to collect and sell or transfer only the oocytes into which the sample has been introduced at a specific position and depth. When selling or transferring, multiple oocytes are packaged and the sample type, introduction date, quality assurance period,
A label 22 describing information such as setting conditions relating to the position and depth at which the sample is introduced and the guaranteed introduction efficiency can be attached (FIG. 3).

Further, according to the present invention, an oocyte can be sold or transferred with information relating to the specific depth and position, the expression of the protein encoded by the introduced gene, and the like. When selling or transferring an oocyte that guarantees sample introduction efficiency and expression efficiency, for example, a protein containing a dye or a chromophore, a fluorescent protein, or a gene encoding these proteins is co-introduced with the sample to give a colored sample. Or count the number of oocytes that emit fluorescence, and use that ratio as an index of sample introduction efficiency,
Sample introduction efficiency can be guaranteed. The co-introduction may be performed in a mixed form. However, when both the sample to be co-introduced and the protein or the like for detection are in the form of a gene, it may be a gene encoding a fusion protein.

An example of calculating the sample introduction efficiency or expression efficiency using the fluorescence from the co-introduced fluorescent protein as an index,
This will be described with reference to FIG. In this example, an example in which the rate of introduction of the histamine receptor gene was tested using the expression of green fluorescent protein (GFP) derived from Oan jellyfish as an index will be described.
The present invention does not limit the sample to be introduced to genes. In addition, substances used as indicators of sample introduction efficiency are also
It is not limited to GFP.

Using a device equipped with the above mechanism or the above principle, a mixture of a histamine receptor gene and a green fluorescent protein gene is introduced into the white surface of an oocyte. When the oocytes 24 hours after the gene transfer operation are irradiated with light having a wavelength of 488 nm, those expressing the green fluorescent protein emit fluorescence of 507 nm. Cells that emitted 507 nm fluorescence were classified as bright, and cells that did not emit fluorescence were classified as dark. Furthermore, these cells were stimulated with histamine and classified according to the presence or absence of a response. FIG. As shown in FIG. 4, in the bright cells, 85% of the cells (34 out of 40) responded to histamine. Ie
The histamine receptor gene has been introduced into more than 85% of the cells. Conversely, in the dark cells, more than 90% of the cells did not respond to histamine (27 of 28). That is, the histamine receptor gene has not been introduced into 90% or more of the cells. In other words, it was found that oocytes expressing the green fluorescent protein had a high proportion of the histamine receptor gene introduced.

As is apparent from this, if the target sample is co-introduced with a fluorescent protein or the like, the efficiency of introducing the target sample can be calculated using the presence or absence of the fluorescence as an index.
Oocytes can be sold or transferred with guaranteed sample introduction efficiency.

Next, using an example using an oocyte into which human histamine receptor cRNA has been introduced, means for producing, selling or transferring the oocyte obtained by the method of the present invention for a specific use However, the gene used for introduction is not limited to cRNA, and may be DNA and RNA, or a synthesized oligonucleotide.

The histamine receptor cRNA is introduced into an oocyte using the above-described apparatus and method according to the present invention. At this time, if a means for obtaining visual information such as a black-and-white discriminating sensor or a CCD camera / digital camera is used, it is possible to discriminate between an oocyte having cRNA introduced on a black surface and an oocyte having cRNA introduced on a white surface.

The histamine receptor is expressed in the oocyte within 24 hours after the gene transfer operation. At least 24 hours after the operation of introducing the histamine receptor gene, the membrane potential of the oocyte in which the histamine receptor is considered to be expressed is fixed at −60 mV by a two-electrode membrane voltage clamp method. Under these conditions, when a sample containing histamine is added to an oocyte, the histamine in the sample and the histamine receptor interact, activating the signaling system in the oocyte, and generating an ion current. Therefore, the oocyte shows an electrical response to histamine. Oocytes after 24 hours are stimulated with 1 μM histamine and the current response is measured. FIG. 5 shows a comparison of the magnitude of the current response of the oocyte into which the gene was introduced on the black or white surface. As shown in FIG. 5, a difference in current response to histamine stimulation was observed depending on the gene transfer site.
That is, it was shown that an oocyte having better ligand responsiveness was obtained when the gene was introduced into the black surface than when the gene was introduced into the white surface.

Therefore, for example, an oocyte population in which the histamine receptor cRNA is introduced only into the black surface can be used as a sensor having high sensitivity to histamine. Further, according to the present invention, an oocyte in which a sample is introduced at a specific position and a specific depth can be sold or transferred for the purpose of using it as a sensor. As will be readily appreciated by those skilled in the art, a sample that can be introduced to use the oocytes obtained according to the present invention as a sensor is the gene for the receptor for any ligand other than histamine, the response to a particular antigen. Antibodies, glycoproteins having specific sugar chains, and the like, and are not particularly limited.

The histamine receptor gene 31 is introduced into the oocyte 13 by using a device having the above mechanism or the above principle. FIG. 6 shows a case where the light is introduced on a white surface for convenience. The histamine receptor 32 is expressed in the oocyte 13 within 24 hours after the histamine receptor gene transfer operation. Similarly to the above, 24 hours or more after the histamine receptor gene introduction operation, the membrane potential of the oocyte in which the histamine receptor 32 is considered to be expressed was determined by the two-electrode membrane voltage-clamp method by −60.
Fix to mV. Under these conditions, oocytes 13
When histamine-containing sample 33 is added to histamine, the histamine in the sample interacts with the histamine receptor, the signal transduction system in the oocyte is activated, and an ion current is generated. An electrical response 34 is shown. When a sample 35 containing no histamine is added, the oocyte 13 does not respond to histamine 36 because there is no substance that interacts with the receptor 36.

That is, a sample is added to an oocyte into which a histamine receptor gene has been introduced, and the presence or absence of histamine in the sample can be sensed using the presence or absence of the cell response as an index. Therefore, by using the sample introduction device of the present invention, a large amount of oocytes having the same sample introduction conditions can be produced, and amphibian oocytes can be produced as a receptor, a ligand that reacts with an antibody, or an antigen. Etc. for screening. That is, screening is carried out by introducing genes and the like under substantially the same conditions, reacting different ligands and the like with each of a plurality of oocytes expressing proteins and the like, and comparing the results. Can be.

As another application, the expressed protein or the like can be extracted by crushing an oocyte expressing the protein or the like, but the introduction conditions can be extracted using the introduction apparatus of the present invention. For example, a desired protein or the like can be efficiently extracted by using, for example, cells having a sample introduced on a black surface.

Next, a method for transporting an oocyte into which a sample according to the present invention has been introduced will be described with reference to FIG. When selling and transporting the above oocytes, a container 21 filled with a solution obtained by adding an antibiotic such as gentamicin sulfate, penicillin, streptomycin to a buffer usually used for amphibian oocytes is used. The oocyte 13 is put therein, and the packing material such as styrofoam is used. While avoiding an impact, as shown in FIG.
C., preferably at 18-22.degree. C. and transported.

The composition of the above solution is not particularly limited, but for example, the following composition can be suitably used. The pH of this solution is 7.5. NaCl 96 mM KCl 2 mM CaCl ₂ 1.8 mM MgCl ₂ 1 mM HEPES 5 mM Gentamicin sulfate 50 μg / ml Sodium pyruvate 2.5 mM penicillin 10 U / ml Streptomycin 10 μg / ml

Suitable containers for sale and transportation include:
There is no particular limitation as long as the cells can be relatively freely moved in the container 21 and can be hermetically sealed with a lid that can be opened and closed, and it is preferable to fill the solution up to about 90% and 5 minutes of its volume. . For example, in the case of a 50 ml conical tube, about 100-200 oocytes, preferably about 130-180
It is preferable to contain individual cells. This ratio corresponds to about 0.3 to 0.5 ml per oocyte, but can vary depending on the type of oocyte, the type of container, and the like.

As shown in FIG. 7, the oocyte 13 into which the sample has been introduced is collected from the tray 9 using a dropper 41 or the like.
At this time, based on the information previously recorded, only the oocytes into which the sample has been introduced on the black surface or the white surface can be collected. The collected oocytes are transferred to the container 21. The container 21 is filled with the amphibian buffer solution 42, and the solution is exchanged several times. After the replacement, the container 21 is filled again with the amphibian buffer solution 42, and an appropriate amount of the antibiotic 43 is further added. Close the lid of the tube and place it in the outer container 44 containing the cold insulator. The packing material 45 and the like are put in the outer container 44, and the container 21 is fixed, and is transported to a supplier by a general transporting method.

According to this method, the oocyte into which the sample has been introduced can be transported to the supplier without impairing the function. At the time of sale / transfer, information such as the introduction date / time, the place where the sample was introduced, the introduction condition such as depth, the recall, the quality assurance period, etc. is also provided. Such methods include, for example, attaching a sheet of paper describing the information, attaching a label 22 to a container 21 having a plurality of oocytes (FIG. 3).

When a gene is introduced, about 2
It takes about 4 hours and the life of the oocyte is 7
Since it is a guideline for about days, more preferably about 5 days, in order to clarify the date and time of introduction and the corresponding effective period after introduction, it is desirable to use up to ○○○○
And so on. About the introduction surface, about 80% of the oocytes can be introduced into the black surface by using the above tray, and the expression efficiency is determined based on the information of the oocytes accumulated in the above device. It is also possible to increase the degree using co-expression.

[0049]

Industrial Applicability According to the present invention, a sample can be introduced into an oocyte of an amphibian such as a frog with high accuracy at a certain depth, and an oocyte with the same introduction efficiency can be obtained quickly and in large quantities. Can be. The quality of the oocyte or the position where the needle is inserted can be stored as information. Further, it is possible to collect only the oocytes into which the sample has been introduced at a specific position and depth obtained by the method of the present invention, or to sell or transfer the cells while guaranteeing the sample introduction efficiency. Further, according to the type of the introduced sample, the use can be specified and sold or transferred.

[Brief description of the drawings]

FIG. 1 shows an apparatus configuration diagram of the present invention.

FIG. 2 shows an example of the shape of a tray used in the present invention.

FIG. 3 shows an example of a container used in the present invention.

FIG. 4 shows the presence or absence of fluorescence from GFP introduced into oocytes,
The correlation of the ligand responsiveness of the histamine receptor expressed by co-introduction is shown.

FIG. 5 shows the relationship between the gene transfer position (black surface or white surface) and ligand responsiveness due to current response.

FIG. 6 shows the use of an oocyte as a histamine sensor.

FIG. 7 shows a method for transporting oocytes after sample introduction.

[Explanation of symbols]

1: control device, 2: control auxiliary device, 3: monitor, 4: introduction needle moving table, 5: introduction device, 6: introduction needle, 7: CCD camera,
8: Digital camera, 9: Tray, 10: Light source, 11: Cartesian carriage, 12: Horizontal carriage, 13: Oocyte, 14: Amphibian saline, 21: Container, 22: Label, 23: Cool Agent, 31: histamine receptor gene, 32: histamine receptor, 33: sample containing histamine, 34: histamine response (with),
35: Histamine-free sample, 36: Histamine response (none), 41: Dropper, 42: Amphibian buffer, 43: Antibiotic, 44: Outer container, 45: Packing material

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12Q 1/02 C12N 15/00 A (72) Inventor Sayuri Nomura 4-6 Kanda Surugadai Chiyoda-ku, Tokyo Stock (72) Inventor Shokichi Matsunami 2520 Akanuma, Hatoyama-cho, Hiki-gun, Saitama Prefecture, Japan Incorporated Company Hitachi, Ltd. (72) Inventor Sakae Saito 2520 Akanuma, Hatoyama-cho, Hiki-gun, Saitama Prefecture F-term (reference) 4B024 AA11 AA20 BA80 DA02 GA12 HA11 4B029 AA09 AA25 BB11 CC03 HA07 4B063 QA01 QA18 QQ20 QR72 QR80 4B065 AA90X AB01 AC14 BA04 CA46

Claims (32)

[Claims] 1. A tray for holding a plurality of amphibian oocytes, an introduction needle for introducing a sample into the plurality of amphibian oocytes, a driving unit for moving a relative position between the tray and the introduction needle, A controller that controls the movement by inputting the depth of the introduction needle with respect to the tray or the amphibian oocyte at the time of introduction, and introducing a sample into the plurality of amphibian oocytes at the depth. A characteristic amphibian oocyte sample introduction device. 2. The amphibian oocyte sample introduction device according to claim 1, wherein the driving unit drives a relative position between the tray and the introduction needle in a three-dimensional direction. 3. The amphibian oocyte sample introduction device according to claim 1, further comprising a visual information acquisition unit for the amphibian oocyte at the time of sample introduction. 4. The amphibian oocyte sample introduction device according to claim 3, wherein the visual information acquisition unit is a camera. 5. The amphibian oocyte according to claim 3, further comprising means for associating visual information of each of said amphibian oocytes obtained by said visual information acquisition section with a position of each cell on said tray. Cell sample introduction device. 6. The amphibian oocyte sample introduction device according to claim 3, further comprising a storage unit for storing the visual information. 7. The amphibian oocyte sample according to claim 1, wherein the tray has a plurality of holes for holding the plurality of amphibian oocytes. Introduction device. 8. The amphibian oocyte sample introduction device according to claim 7, wherein the hole has a flat bottom surface or a cylinder having a flat bottom surface, and has a maximum diameter of 1.4-2 mm. 9. A cylinder having a flat bottom surface or a conical bottom portion with a maximum diameter of one of the diameters of the plurality of amphibian oocytes.
The amphibian oocyte sample introduction device according to claim 7, wherein the amount is from 0.05 to 150%. 10. The surface position of the oocyte on the tray is represented by visual information, pressure change, temperature change, electrical change, humidity change,
The amphibian oocyte sample introduction device according to any one of claims 1 to 9, wherein the detection is performed by at least one of pH changes. 11. A tray for holding a plurality of amphibian oocytes, an introduction needle for introducing a sample into the plurality of amphibian oocytes, a driving unit for moving a relative position between the tray and the introduction needle, A control unit for controlling movement, an information acquisition unit for obtaining visual information of the amphibian oocyte at the time of introduction, and a storage unit for storing the information, and introducing the sample into the plurality of amphibian oocytes An amphibian oocyte sample introduction system, comprising: 12. The amphibian oocyte sample introduction system according to claim 11, wherein the tray has a plurality of holes for holding the plurality of amphibian oocytes. 13. The method of claim 12, wherein the bottom surface of the hole is a flat cylinder or a conical bottom and the maximum diameter is 105-150% of the diameter of the plurality of amphibian oocytes.
3. The amphibian oocyte sample introduction system according to claim 1. 14. An introduction needle for said tray or said amphibian oocyte, using a device having a tray for holding a plurality of amphibian oocytes and an introduction needle for introducing a sample into said plurality of amphibian oocytes. Setting the depth of the first to a first depth, the first of the plurality of amphibian oocytes
Introducing the sample into the oocyte with the introduction needle to the first depth, automatically moving the relative position of the tray and the introduction needle, and subsequently the plurality of amphibian oocytes Introducing a sample into the second depth of the first oocyte with the introduction needle to the first depth. 15. An apparatus comprising: a tray for holding a plurality of amphibian oocytes; and an introduction needle for introducing a sample into the plurality of amphibian oocytes, wherein a first one of the plurality of amphibian oocytes is used. Introducing a sample into the oocyte of the introduction needle, the step of moving the relative position of the tray and the introduction needle, and then the second of the plurality of amphibian oocytes
Introducing a sample into the oocyte with the introduction needle, obtaining the state of the oocyte at the time of sample introduction as visual information, and accumulating the visual information. , An amphibian oocyte sample introduction method. 16. The method for introducing an amphibian oocyte sample according to claim 14, wherein the sample is a gene or a protein. 17. The method of introducing an amphibian oocyte sample according to claim 14, wherein the sample contains a fluorescent substance. 18. The method according to claim 18, wherein the amount of the sample introduced into the first oocyte and the amount of the sample introduced into the second oocyte are controlled to be substantially the same. 18. The method for introducing an amphibian oocyte sample according to any one of 14 to 17 above. 19. A plurality of amphibian oocytes, wherein each of the samples is introduced under a condition that the introduction depths of the samples are substantially equal. 20. The amphibian oocyte according to claim 19, wherein the sample is introduced under substantially the same conditions at the introduction position. 21. The plurality of amphibian oocytes according to claim 19, wherein the amount of the sample introduced is substantially constant. 22. A plurality of amphibian oocytes, wherein the sample is introduced under the condition that the sample introduction depths are substantially equal, wherein information on the date and time of the introduction and the egg A container to which information on the expiration date of the mother cell is attached. 23. The method according to claim 23, wherein at least one of the sample introduction position and the sample introduction amount has the plurality of amphibian oocytes introduced therein under substantially the same conditions. 23. The container of claim 22, wherein 24. A device having a tray for holding a plurality of amphibian oocytes and an introduction needle for introducing a sample into the plurality of amphibian oocytes, and moving the introduction needle relative to the tray. While allowing the sample to be introduced into each of the plurality of amphibian oocytes with the introduction needle, to obtain visual information of each of the plurality of amphibian oocytes at the time of the introduction,
Introducing among the plurality of amphibian oocytes based on the visual information, wherein collecting a plurality of oocytes introduced on the black surface of the oocyte, the amphibian oocyte into which the sample is introduced Manufacturing method. 25. A device having a tray for holding a plurality of amphibian oocytes and an introduction needle for introducing a sample into the plurality of amphibian oocytes, and moving a relative position of the introduction needle with respect to the tray. While allowing the sample to be introduced into each of the plurality of amphibian oocytes with the introduction needle, to obtain visual information of each of the plurality of amphibian oocytes at the time of the introduction,
Of the plurality of amphibian oocytes based on the visual information, characterized in that the introduction is to collect a plurality of oocytes introduced to the white surface of the oocyte, the sample is introduced amphibian oocyte Production method. 26. The method according to claim 26, wherein the depth of the introduction needle relative to the amphibian oocyte or the tray is set to a first depth before the introduction, and the plurality of amphibian oocytes are sampled at the first depth. 26. The method for producing an amphibian oocyte group according to claim 24, wherein the method is performed. 27. A method for selling or transferring a plurality of amphibian oocytes, wherein a plurality of amphibian oocytes into which a sample has been introduced are set under conditions where the sample introduction depths are substantially equal. . 28. The method for selling or transferring a plurality of amphibian oocytes according to claim 27, wherein information on sample introduction into the plurality of amphibian oocytes is attached to the set. 29. A plurality of amphibian oocytes, each of which has been introduced under the condition that the sample introduction depths are substantially equal, are placed in a container, and information on the sample introduction into the plurality of amphibian oocytes is written in the container. A method of selling or transferring a plurality of amphibian oocytes with a label attached. 30. The method for selling or transferring a plurality of amphibian oocytes according to claim 29, wherein the temperature of the container is adjusted to 18 ° C. or more and 22 ° C. or less. 31. The information on sample introduction into a plurality of amphibian oocytes may be at least one of the date and time of introduction, the quality assurance period of the oocyte, the position at which the sample was introduced, the depth at which the sample was introduced, and the probability of expression. 31. The method for selling or transferring a plurality of amphibian oocytes according to any one of claims 27 to 30, characterized in that it relates to one. 32. A method of using a plurality of amphibian oocytes into which a gene or protein has been introduced at a substantially constant position and depth as a sensor for screening.