JPH09187278A - Introduction of foreign gene into eucaryotic cell and cell holder - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently transduce a foreign gene into an eukaryotic cell by enabling the eukaryotic cell to be held simply and stably, when the foreign gene is transfected into the eukaryotic cell. SOLUTION: A foreign gene accompanying an intranuclear transport protein is transfected into the cytoplasm of an eukaryotic cell. At this time, a cell holder 2 equipped with a recessed part 6 which can receive the eukaryotic cell and having a sucking hole 8 bored on the recessed part 6 is used, as the inside of the recessed part 6 is sucked to tightly hold the cell to the inner surface 6b for transfection of the foreign gene.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for introducing a foreign gene into a eukaryotic cell and a cell holding device.

[0002]

2. Description of the Related Art Conventionally, the microinjection method has been widely used for introducing a foreign gene into a eukaryotic cell, particularly for introducing a foreign gene into a fertilized egg. As shown in FIG. 4, the microinjection method uses a micromanipulator under a microscope to obtain fertilized eggs 5 after meiosis.
0 is held by a holding pipette 52, and a glass pipette 54 for injection having a diameter of about 1 μm is used for the fertilized egg 50.
This is a method in which the female pronucleus 56 or the male pronucleus 58 is reached and the solution 60 containing the foreign gene is injected into the pronucleus. According to this method, the injected foreign gene is integrated into the chromosome of the fertilized egg 50 by gene recombination in the male pronucleus or the like.

[0003]

However, the operation of holding the fertilized egg 50 with the holding pipette 52,
Both the operation of injecting the foreign gene solution 60 with the injection pipette 54 must be performed simultaneously with one hand. Also, these are fine operations under the microscope.
Therefore, it is difficult to say that the microinjection method requires a high degree of skill and can be performed by anyone. Further, in the microinjection method, since such a complicated operation is performed for each fertilized egg, it is impossible to process a large number of fertilized eggs at one time.
Therefore, in order to introduce the foreign gene into the eukaryotic cell by the microinjection method, it has been desired to simplify the holding operation and the injection operation.

Further, as shown in FIG. 5, particularly in fertilized eggs 70 such as pigs and cattle, since many lipid droplets 72 are present in the cells, the pronucleus 74, 76 inside the fertilized eggs 70 is observed under a microscope. Invisible. Therefore, as shown in FIG.
0 is subjected to centrifugation to bring the lipid droplet 72 to one side in the cell to make the visible range as wide as possible. In the presence of the lipid droplet 72, the pronucleus 74, 76 is present.
To adjust the holding position of the fertilized egg 70 so that
It was very difficult. Therefore, in such a fertilized egg 70, it was very difficult to inject a foreign gene into the nucleus.

Therefore, according to the present invention, when a foreign gene is injected into a eukaryotic cell, the operation of holding the eukaryotic cell is simplified to easily and stably hold the eukaryotic cell.
A first object is to provide a method for easily introducing a foreign gene into a eukaryotic cell. Further, in the present invention, by injecting a foreign gene into the cytoplasm of a eukaryotic cell to allow introduction of the foreign gene into the nucleus, the injection operation of the foreign gene into the nucleus of a eukaryotic cell is simplified. A second object is to provide a method for easily introducing a foreign gene into a eukaryotic cell. A third object of the present invention is to provide a device that can easily and stably hold cells.

[0006]

In order to solve the above technical problems, the present inventor has completed the following inventions. That is, a first invention for solving the above-mentioned first problem is a method for introducing a foreign gene into a eukaryotic cell, which comprises a recess capable of accommodating a eukaryotic cell and a suction hole formed in this recess. A foreign gene to a eukaryotic cell, characterized by injecting a foreign gene into a eukaryotic cell that is tightly held on the inner surface of the recess by sucking the inside of the recess from the suction hole using a cell holding device comprising It is an introduction method. According to the present invention, by sucking the concave portion from the suction hole, the eukaryotic cell is stably sucked and held in a state of being closely attached to the inner surface of the concave portion. Since the eukaryotic cell is in close contact with the inner surface of the recess, the eukaryotic cell is stably held at a fixed position without being shaken or rotated. That is, the operation for holding eukaryotic cells is simplified as compared with the conventional method. In addition, when a foreign gene is injected into the thus-retained eukaryotic cell by the microinjection method, the eukaryotic cell is stably retained even when it is inserted by a pipette for injection. Therefore, the foreign gene is simply and reliably injected into a desired site in the cell. Also, a large number of processes can be performed in a relatively short time and more efficiently than in the conventional technique.

A second invention for solving the second problem is a method for introducing a foreign gene into a eukaryotic cell, the foreign gene being accompanied by a nuclear transport protein, the cytoplasm of the eukaryotic cell. It is a method for introducing a foreign gene into a eukaryotic cell, which is characterized in that it is injected inside. Nuclear transport proteins are proteins that are transported from the cytoplasm into the nucleus. That is, according to this method, along with the nuclear transport protein,
Foreign genes can be transferred into the nucleus. Therefore, the complicated operation of injecting the foreign gene into the nucleus is omitted, and the foreign gene is easily introduced into the nucleus. In the present invention, the nuclear transport protein is not limited to the nuclear transport protein possessed by a eukaryotic cell into which a foreign gene is to be introduced, but targets the nucleus of a eukaryotic cell into which a foreign gene is to be introduced. Is a protein that can be transported as. Therefore, it may be a nuclear protein of a eukaryotic cell or virus of a species different from the eukaryotic cell into which the foreign gene is introduced.

Further, according to the present invention, a cell holding device having a recess capable of accommodating a eukaryotic cell and a suction hole formed in the recess is used, and the inside of the recess is sucked by sucking the inside of the recess from the suction hole. It is preferable to inject a foreign gene into the cytoplasm of a eukaryotic cell that is held in close contact with the surface. According to this method, the operation of injecting into the nucleus is omitted, and the eukaryotic cell is easily and stably retained, and the operation of retaining the eukaryotic cell is simplified. Therefore, the foreign gene and the nuclear transport protein are efficiently injected into the cytoplasm of the eukaryotic cell using an injection pipette or the like. In addition, it becomes easy to inject the foreign gene and the nuclear transport protein into the vicinity of the nucleus so that the foreign gene can be rapidly transferred into the nucleus.

Further, a third method for solving the second problem
The present invention is a method for introducing a foreign gene into a eukaryotic cell, which comprises injecting into the cytoplasm of the eukaryotic cell by applying a voltage with an anode and a cathode inside the cytoplasm of the eukaryotic cell. Is a method for introducing a foreign gene into a eukaryotic cell, which is characterized in that the foreign gene is transferred into the nucleus. According to this method, the stability of the nuclear membrane is changed by the application of a voltage, and the foreign gene injected into the cytoplasm is easily transferred into the nucleus. In addition, by applying a voltage, the foreign gene as a charged substance can be given a direction of movement in the cytoplasm, and the foreign gene can be directed to the nucleus and moved in the cytoplasm. Therefore, by applying a voltage, the foreign gene injected into the cytoplasm is transferred into the nucleus. As a result, the cumbersome operation of injecting the foreign gene into the nucleus is omitted, and the foreign gene is easily introduced into the eukaryotic cell.

Further, according to the present invention, a cell holding device provided with a recess capable of accommodating a eukaryotic cell and a suction hole formed in the recess is used, and the inside of the recess is sucked by sucking the inside of the recess from the suction hole. It is preferable to inject a foreign gene into the cytoplasm of a eukaryotic cell that is held in close contact with the surface. According to this method, the operation of injecting a foreign gene into the nucleus is omitted, and further, the operation of holding the eukaryotic cell is simplified and the eukaryotic cell can be stably held. Therefore, the internal operation of the electrode by needle insertion or the like can be easily performed, and the foreign gene can be injected or the electrode can be internalized at a desired site. As a result, the foreign gene is easily introduced into the eukaryotic cell. In particular, if a foreign gene is injected into the vicinity of the nucleus, the foreign gene can be more easily introduced into the nucleus.

In the present invention, the foreign gene is
It is preferable to inject it into the cytoplasm of the eukaryotic cell together with the nuclear transport protein. According to this method, by applying a voltage, the operation of injecting a foreign gene into the nucleus is omitted, and further, the injected foreign gene is easily transferred into the nucleus by the nuclear transport protein. Therefore,
More efficiently, the foreign gene is easily introduced into the nucleus.

Further, according to the present invention, a cell holding device having a recess capable of accommodating a eukaryotic cell and a suction hole formed in the recess is used, and the inside of the recess is sucked by sucking the inside of the recess from the suction hole. It is preferable to inject the foreign gene into the cytoplasm of the eukaryotic cell that is held in close contact with the surface together with the nuclear transport protein of the eukaryotic cell. According to this method,
Simplification of cell retention operation by stable retention of eukaryotic cells, omission of intranuclear injection operation by injection of foreign gene with nuclear transport protein, change of stability of nuclear membrane by application of voltage and injection material (foreign material Omission of the intranuclear injection operation by imparting the direction of movement of the gene and the nuclear transport protein) in the cytoplasm facilitates the introduction of the foreign gene into the nucleus.

Further, the above-mentioned first invention, second invention,
In the third invention, the eukaryotic cell is preferably a fertilized egg cell. This is because the microinjection method was the only effective method for introducing a physical foreign gene into fertilized egg cells. That is, when the first invention is applied to fertilized egg cells, the complicated holding operation of the conventional microinjection method is simplified, and the microinjection method for fertilized eggs can be easily and efficiently performed. In particular, in a fertilized egg in which it is difficult to visually recognize the pronucleus due to the presence of fat droplets, it becomes easy to hold the pronucleus under a microscope so that an efficient injection operation can be performed. When the second or third invention is applied to a fertilized egg cell, the intranuclear injection operation is omitted, and the foreign gene is introduced into the nucleus of the fertilized egg more easily. This embodiment is particularly preferable when the fertilized egg cell contains lipid droplets and it is difficult to visually recognize the pronucleus under a microscope. This is because according to this aspect, it is not necessary to visually recognize the pronucleus. Furthermore, when the first and second inventions, or the first and third inventions are applied to fertilized egg cells, the conventional complicated holding operation in the microinjection method of fertilized eggs is simplified. The foreign gene is introduced into the nucleus of a fertilized egg more easily by omitting the intranuclear injection operation. This embodiment is particularly preferable when the fertilized egg cell contains lipid droplets and it is difficult to visually recognize the pronucleus under a microscope. This is because according to this aspect, it is not necessary to visually recognize the pronucleus.

A fourth invention for solving the above-mentioned third problem has a recess capable of accommodating the eukaryotic cell, and a suction hole formed in this recess and capable of sucking the inside of the recess. A cell holding device, characterized in that the inside of the concave portion is sucked through the suction hole, and the cells accommodated in the concave portion are held by being brought into close contact with the inner surface of the concave portion. According to the present invention, by sucking the concave portion from the suction hole, the cells are stably sucked and held in a state of being brought into close contact with the inner surface of the concave portion. Since the eukaryotic cell is in close contact with the inner surface of the recess, the eukaryotic cell is stably held at a fixed position without being shaken or rotated. Further, even if the cells thus retained are subjected to a nucleus removal operation or a gene injection operation with a pipette, a cutter or the like, the cells are stably retained. It is preferable to apply this cell holding device to fertilized egg cells. In various operations on fertilized eggs,
This is because it has been difficult to hold fertilized eggs in the past.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION Next, a detailed description will be given based on an embodiment of the present invention. In the present invention, injecting a foreign gene into a eukaryotic cell means injecting a solution containing the foreign gene into the cytoplasm or nucleus of the eukaryotic cell. Introducing a foreign gene into a eukaryotic cell means injecting the foreign gene into the eukaryotic cell so that the foreign gene is internally present in the nucleus of the eukaryotic cell.

The cell holding device of the first invention and the fourth invention is a eukaryotic cell or cell (hereinafter abbreviated as eukaryotic cell etc.).
And a suction hole formed in this recess. The concave portion capable of accommodating a eukaryotic cell or the like is a concave portion having an opening through which one eukaryotic cell or the like can enter and having a size and a shape capable of holding one eukaryotic cell or the like. It is preferably a recess having an inner surface along a region of about 1/3 of the surface of eukaryotic cells or the like. Furthermore, it is preferably a recess having an inner surface along a region of about 1/2 of the surface of the eukaryotic cell or the like. As the specific shape of the concave portion, a substantially hemispherical shape, a truncated cone shape, a truncated pyramid shape, a cone shape, a pyramid shape,
Examples include a cubic shape and a rectangular parallelepiped shape.

At least one suction hole is provided inside the recess.
Although it may be provided at a plurality of locations, it may be provided at a plurality of locations so as to facilitate holding of eukaryotic cells and the like.
The suction hole is communicated with a suction device provided separately or integrally with the cell holding device. Examples of the suction device include a syringe for injection. In the case of a syringe for injection, liquid is contained in the cylinder, and by operating the piston, the liquid is sucked and discharged from the tip of the syringe, and along with this, the liquid filling the suction hole and recess is also sucked. As a result, the inside of the recess can be sucked through the suction hole. The suction mechanism in the suction hole is not limited to this, and another known suction mechanism can be used.

By sucking the inside of the concave portion from the suction hole,
To inject a foreign gene into a eukaryotic cell that is held in close contact with the inner surface of the recess, an injection pipette used for microinjection can be used.
Other alternatives can also be used.

The material for the cell holding device is not particularly limited. However, even if the cells are fertilized eggs, the diameter is 10
Since it is about 0 μm, a fine processing technique is required for forming the recess and the suction hole. Therefore, it is preferable to form the cell holding device by using various semiconductor substrates, glass substrates and the like used in the semiconductor integrated circuit manufacturing process and using the lithography technique and the etching technique used in the same process. At the time of use, this cell holding device is in a state of being entirely immersed in a culture solution or buffer solution of eukaryotic cells or the like. Therefore, the concave portion and the suction hole are also filled with the culture solution or the like. In order to suck the inside of the concave portion from the suction hole, it is preferable to interpose a sufficient amount of the culture solution or the like. This is because in order to aspirate and hold eukaryotic cells and the like, it is necessary to finely adjust the suction pressure, and it is difficult to finely adjust the suction pressure by suction with a small amount of culture solution or the like.

As the eukaryotic cell and the like in the present invention, it is particularly useful to use a fertilized egg cell, but particularly for bovine or porcine cells in which it is difficult to confirm the pronucleus in the fertilized egg by a lipid droplet under a microscope. It is more preferable to apply the present invention to the introduction of a gene into a fertilized egg. According to the present invention, it is easy to stably hold the fertilized egg and to easily hold and adjust the pronucleus under a microscope.

Further, in the second invention, in order to inject the foreign gene into the cytoplasm of the eukaryotic cell together with the nuclear transport protein, a mixed solution of the foreign gene and the nuclear transport protein or a nuclear transport protein is used. It can be carried out by injecting into the cytoplasm a solution of a conjugate of a protein and a foreign gene, a solution containing a part or the whole of the foreign gene in the nuclear transport protein, and the like. The state of binding between the nuclear transport protein and the foreign gene or the inclusion state of the latter by the former can be selected as necessary.
For example, examples of the bond between the nuclear transport protein and the foreign gene include a hydrogen bond, an electrostatic bond, a covalent bond such as a disulfide bond, and the like. In addition, the inclusion state of the foreign gene by the nuclear transport protein forms a site that becomes a signal when the nuclear transport protein passes through the nuclear pore,
Alternatively, the foreign gene is included so that it is exposed.

Examples of the nuclear transport protein include a nuclear protein of a eukaryotic cell and a protein of a virus that infects a eukaryotic cell. These nuclear proteins are proteins that function in the nucleus of eukaryotic cells and pass through the nuclear pore to enter the nucleus. Nuclear proteins have a specific amino acid sequence in their molecule that serves as a signal for localization in the nucleus. That is, this amino acid sequence has the activity of bringing into the nucleus a molecule that is not originally localized in the nucleus, and the nuclear localization signal (nucl
ear loca1ization signal: N
It is said to be LS). Therefore, the term “nuclear transport protein” as used in the present invention means not only a nuclear protein of a eukaryotic cell and a protein of a virus that infects a eukaryotic cell but also NLS of such a protein. Examples of the protein having NLS include SV40T antigen and nucleoplasmin of various eukaryotic cells including Xenopus oocytes and these have been cloned (C. Dirgwall and R. et al.
Laskey, 1986, Ann. Rev .. CellB
iol. 2: 367-390, R.M. H. Lyons,
B. Furguson, M .; Rosenhey, 198
7, Mol. Cell Biol. , 7: 2451-2
456). In addition, influenza virus nucleoprotein (NP), adenovirus Ela, yeast m
Atα2, ribosomal protein L3 and the like are known.

In the third invention, in order to make the electrode internal in the cytoplasm of the eukaryotic cell, for example, the electrode can be pierced from the outside of the cell into the cytoplasm. The material of the electrode is
Although it can be used without particular limitation, one of the cathode and the anode can also serve as a pipette for injecting the foreign gene solution. In this case, a voltage can be applied at the same time as the injection of the foreign gene solution using the electrode / pipette. The electrodes are preferably internal with the tip near the nucleus. More preferably, it is made internal in the state of being in contact with the nuclear membrane. In general, negatively charged DNA moves to the anode side when a voltage is applied. Therefore, it is preferable to have the cathode and the anode internal so that the foreign gene can move toward the nucleus in the cytoplasm.

The embodiments of the present invention will be specifically described below. It should be noted that the present invention is by no means limited to the following examples. (Example) In this example, a method for introducing a foreign gene into a fertilized egg P of a pig will be described by way of example. FIG. 1 shows a plan view and a sectional view of a device 2 for this embodiment. In this embodiment, the device 2 is made of glass and has a substrate 4 having a size of about 1.5 mm × 0.5 mm.
The suction channel 10 and a suction device (not shown) are formed. When used, the device 2 is used by being immersed in the culture solution of the fertilized egg P as a whole. The base 4 has a total of 16 hemispherical recesses 6 having a diameter of about 80 μm and a depth of about 40 μm, arranged in two rows and eight rows. The recess 6 is formed to have a size substantially corresponding to half of the fertilized egg P of the pig, and the fertilized egg P of the pig can be easily housed in the recess 6 and the fertilized egg stored therein. P
About half of the above is exposed upward from the opening 6a of the recess 6.

One suction hole 8 is opened at the bottom of each recess 6. The suction hole 8 penetrates the substrate 4 downward and communicates with a suction channel 10 provided on the lower side of the substrate 4. The suction flow path 10 is filled with the culture solution, and a part thereof is opened and connected to the suction device provided outside through the culture solution. When the device 2 is used, the suction device is operated to suck or discharge the culture solution filled in the suction channel 10, and the culture solution filled in the suction channel 10 is sucked or discharged through the suction channel 10 and the suction hole 8. The fertilized egg P inside can be suction-held.

In order to introduce a foreign gene into the fertilized egg P using this apparatus 2, a glass cathode 12 and a glass anode 14 are also used. In particular, the cathode 12 has a through hole 12a formed inside along the length direction,
The through hole 12a also serves as a pipette for injecting the foreign gene solution into the cytoplasm at the same time (see FIG. 3). Each of these electrodes 12 and 14 is connected to a power source provided outside the device 2, and a voltage is applied thereto.

In this example, the foreign gene to be introduced into the fertilized egg P is used in a state in which it is bound to the nuclear transport protein Xenopus laevis nucleoplasmin by a disulfide bond. That is, cysteine is bound to the 3'-end or 5'-end of the foreign gene, and a disulfide bond is formed between this cysteine and cysteine of nucleoplasmin. Foreign gene-nucleoplasmin complex 2 thus formed
6 is introduced into the through hole 12a in the cathode 12 in a solution state. In this embodiment, the complex 26 is formed by the disulfide bond, but the complex may be formed by hydrogen bond, electrostatic bond or the like. Also, in this example, the foreign gene is
The neomycin resistance gene was used.

Next, the process of introducing a foreign gene into the fertilized egg P after meiosis using this device 2 will be described. FIG. 2 shows a state in which the device 2 is placed on the plate 16 under the microscope of the micromanipulator. A shallow recess 18 is formed in the center of the plate 16, and the device 2 is placed in the center of the recess 18. The suction channel 10 of the device 2 is filled with the culture solution, and the culture solution inside the recess 6 can be sucked and discharged through the opening formed in the side portion of the base 4. It is in the state of being connected to the suction device.
In this state, a plate containing the culture medium containing fertilized egg P
When the recesses 18 of 6 are filled, the fertilized egg P is set in the recesses 6 of the device 2 due to gravity. Then, a paraffin oil film is applied so as to cover the surface of the culture solution in the recess 18.

Then, by operating a suction device (not shown),
The fertilized egg P in the recess 6 is sucked through the suction hole 8 to adjust the holding of the fertilized egg P appropriately. As a result, as shown in FIG. 3, the fertilized egg P is held along the inner side surface 6b of the recess 6 and partially held in close contact with the inner surface 6b to be stably held. . In this holding state, the fertilized egg P is in a state in which the fat droplets 20 are dispersed in the cytoplasm, and the pronuclei 22 and 24 in the cytoplasm are invisible under the microscope.

Next, the cathode 12 and the anode 14 are connected to the fertilized egg P.
The transparent body and the cell membrane are penetrated, and the tip side of each is made to be internal in the cytoplasm. The respective tips are positioned opposite to each other with the male pronucleus 24 in between. In particular, the cathode 12, which also serves as an injection pipette, is located near the male pronucleus 24.
At this time, since the fertilized egg P is stably held in the recess 6 by the suction through the suction hole 8 and the close contact with the inner surface 6b of the recess 6, when the cathode 12 and the anode 14 are pierced into the cytoplasm. Also, the fixed state is maintained. For this reason, the electrodes 12 and 16 can be reliably made to exist in the cytoplasm.

Further, a solution containing the foreign gene-nucleoplasmin complex 26 is injected into the cytoplasm through the through hole 12a of the cathode 12. At the same time, these electrodes 12, 1
A voltage is applied between the four. Application of voltage and the composite 26
Of the foreign gene-nucleoplasmin complex 26, which has flowed out from the tip of the cathode 12, due to the outflow of
Move to the anode 14 side. Then, a part of the foreign gene-nucleoplasmin complex 26 passes through the nuclear pore of the male pronucleus 24 and is transferred into the male pronucleus 24. The complex 26 after being transferred into the pronucleus 24 is decomposed by cleavage of the disulfide bond in the nucleus, and is separated into a foreign gene and nucleoplasmin. Less than,
The above operation is repeated for the fertilized eggs P stored in the other recesses 6.

In this example, the foreign gene is in a state of being easily passed through the nuclear membrane of the male pronucleus 24 because it is in a complex with the nuclear transport protein nucleoplasmin. Moreover, the application of voltage imparts directionality to the movement of the complex in the cytoplasm. Furthermore, since the voltage is applied, the stability of the nuclear membrane of the male pronucleus 24 is changed, and it is easy for the nuclear membrane of the male pronucleus 24 to pass through.

Further, in particular, in this embodiment, the pronucleus 2
Even in the fertilized egg P in which 2 and 24 are not visible, if the solution containing the foreign gene is injected into the cytoplasm, the female pronucleus 22
Alternatively, a foreign gene can be introduced into the male pronucleus 24. Therefore, the foreign gene can be introduced into the nucleus by a simple operation without directly injecting the foreign gene into the male pronucleus 24. Further, in a fertilized bovine egg in which the pronucleus cannot be visually recognized like the fertilized egg P of pig, according to the present example, a foreign gene can be introduced into the nucleus by a simple operation.

In this embodiment, once the fertilized egg P is stably held in the concave portion 6 by the present device 2, the needle 12 is inserted into the concave portion 6 at a fixed position.
A solution of a predetermined amount of foreign gene-nucleoplasmin complex 26 is formed so as to be ejectable from the through hole 12a of the cathode 12,
Furthermore, by moving either or both of the device 2 and the electrodes 12 and 14, it is possible to automatically drive the gene transfer operation to each fertilized egg P. According to such an automatic operation, since many fertilized eggs can be processed without manpower, labor saving of foreign gene introduction operation into eukaryotic cells can be further achieved.

[Brief description of the drawings]

FIG. 1 is a plan view (a) and a sectional view (b) of a cell holding device.
It is.

FIG. 2 is a diagram showing a state in which a cell holding device is placed on a plate in a micromanipulator.

[Fig. 3] Sucking and holding a fertilized egg in the recess of the cell holding device,
It is a figure which shows the state which injected the foreign gene-nucleoplasmin complex solution into the cytoplasm.

FIG. 4 is a diagram showing an operation of injecting a foreign gene into the nucleus of a fertilized egg by the microinjection method.

FIG. 5 is a diagram showing a fertilized egg in which lipid droplets are dispersed in the cytoplasm.

FIG. 6 is a view showing a state in which a fertilized egg is held by a holding pipette and a pronucleus is visually recognized in a state where fat droplets are unevenly distributed by centrifugation.

[Explanation of symbols]

 2 Cell Retaining Device 6 Recess 6a Recess Inner Surface 8 Suction Hole 12 Cathode 14 Anode

Claims (10)

[Claims] 1. A method for introducing a foreign gene into a eukaryotic cell, which comprises using a cell holding device having a recess capable of accommodating the eukaryotic cell and a suction hole formed in this recess A method for introducing a foreign gene into a eukaryotic cell, which comprises injecting a foreign gene into a eukaryotic cell that is held in close contact with the inner surface of the recess by suctioning the inside of the recess from the cell. 2. A method for introducing a foreign gene into a eukaryotic cell, which comprises injecting the foreign gene into the cytoplasm of the eukaryotic cell together with a nuclear transport protein. Foreign gene transfer method. 3. A method for introducing a foreign gene into a eukaryotic cell, wherein an anode and a cathode are made to exist inside the cytoplasm of the eukaryotic cell and a voltage is applied to the cytoplasm of the eukaryotic cell. A method for introducing a foreign gene into a eukaryotic cell, which comprises transferring the injected foreign gene into the nucleus. 4. The method for introducing a foreign gene according to claim 2, wherein the cell holding device is provided with a recess capable of accommodating the eukaryotic cell and a suction hole formed in the recess. A method for introducing a foreign gene into a eukaryotic cell, which comprises injecting the foreign gene into the cytoplasm of a eukaryotic cell that is tightly held on the inner surface of the recess by sucking the inside of the recess from the cell. 5. The method for introducing a foreign gene according to claim 3, wherein the cell holding device is provided with a recess capable of accommodating the eukaryotic cell and a suction hole formed in the recess. A method for introducing a foreign gene into a eukaryotic cell, which comprises injecting the foreign gene into the cytoplasm of a eukaryotic cell that is tightly held on the inner surface of the recess by sucking the inside of the recess from the cell. 6. The method for introducing a foreign gene according to claim 3, wherein the foreign gene is injected into the cytoplasm of the eukaryotic cell together with a nuclear transport protein. Method for introducing foreign gene into cells. 7. The method for introducing a foreign gene according to claim 6, wherein an anode and a cathode are made to exist in the cytoplasm of the eukaryotic cell and a voltage is applied to the cytoplasm of the eukaryotic cell. A method for introducing a foreign gene into a eukaryotic cell, which comprises transferring the injected foreign gene into the nucleus. 8. The method for introducing a foreign gene according to claim 1, 2, 3, 4, 5, 6 or 7, wherein the eukaryotic cell is a fertilized egg cell. Foreign gene transfer method. 9. A recess for accommodating cells, and a suction hole formed in the recess for suctioning the inside of the recess, wherein the suction hole sucks the inside of the recess into the recess. A cell holding device, which holds the arranged cells by bringing them into close contact with the inner surface of the recess. 10. The cell holding device according to claim 9, wherein the cells are fertilized egg cells.