JP2007089426A - Gene introduction device and gene introduction method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To surely introduce genes prepared for transformation, transduction, or the like into target cells in a large amount in simple processes. <P>SOLUTION: This gene introduction device for introducing genes into target cells comprises at least the first electrode 142 connected to the cathode of an electric source 112, the first buffer solution-holding portion 143 disposed on the front side of the first electrode 142 and holding a buffer solution, the first cation exchange membrane 144 disposed on the further front side of the first buffer solution-holding portion 143, a gene-holding portion 145 disposed on the further front side of the first cation exchange membrane 144 and holding the genes, and the first anion exchange membrane 146 disposed on the further front side of the gene-holding portion 145. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a gene transfer apparatus, and more particularly to a gene transfer apparatus and a gene transfer method for the purpose of transformation of a living organism or transduction into an organism.

  Conventionally, various gene introduction apparatuses for introducing foreign genes into cells for the purpose of transduction for introducing new traits into organisms or transformation for transforming inherent traits of organisms, Gene transfer methods are known.

  For example, a method using calcium phosphate (hereinafter sometimes simply referred to as “calcium phosphate method”) is known. In this method, a calcium ion having a positive charge is bound to a gene having a negative charge, and phosphoric acid is added thereto, whereby the calcium ion and the phosphate are bound to cause precipitation of the complex. This is a method of introducing a foreign gene into a cell by incorporating the calcium phosphate / gene complex into the cell by endocytosis, which is a function inherent to the cell.

  In addition, lipid bilayer vesicles (liposomes) composed of positively charged lipids and the like are electrically interacted with the gene to be introduced to form a complex, resulting in cell phagocytosis and membrane fusion. There is also a method (hereinafter sometimes simply referred to as “lipofection method”).

  In addition, by applying a high-voltage pulse to cells, the cell membrane structure of the lipid bilayer is temporarily destabilized and a foreign gene is incorporated (hereinafter simply referred to as “electroporation method”). .)

  In addition, a method of directly introducing a foreign gene into individual cells using a very fine glass injection needle (hereinafter sometimes simply referred to as “microinjection method”) is also known.

  Also, a method of introducing a target gene into a target cell by incorporating the gene to be introduced into the attenuated virus and infecting the target cell with the virus (hereinafter simply referred to as “virus vector method”). There are cases.)

  Furthermore, an apparatus as described in Patent Document 1 is also known. The apparatus described in this patent document 1 is shown in FIG.

  The gene introduction apparatus described in Patent Document 1 includes a DNA solution in which a plasmid (gene) to be introduced is dissolved inside a pipette tip 34, and negative electricity is applied to the solution by an electrode 36. ing. The gene DNA (“plasmid” in FIG. 6 corresponds to this gene) is charged to a negative charge as a whole gene by its own phosphate group. For this reason, when a negative charge is applied from the electrode 36, the gene is moved into the cell or inside the nucleus, that is, into the anode side, and is to be introduced.

JP 2004-321136 A

  However, the above-mentioned calcium phosphate method has the merit that it can be carried out relatively easily without the need for special equipment or technology, but passively takes up the calcium phosphate-gene complex by endocytosis. The gene transfer efficiency is not necessarily high.

  In the lipofection method, transfection efficiency is high, and various genes such as oligonucleotides, high molecular DNA, messenger RNA, single-stranded RNA can be introduced, and the operation is completed in a short time. Many samples can be processed, and there is an advantage that no special apparatus or equipment is required. However, there are disadvantages in that the optimal conditions for transfection (cell density, gene amount, liposome amount, culture time, medium, etc.) are very narrow and the reagents used are very expensive.

  In addition, according to the electroporation method, although it is possible to introduce a gene with a simple operation and a high probability, the apparatus itself is expensive, and in particular, for each kind of cell and each kind of gene to be introduced. Since the voltage, electric pulse length, temperature, cell and gene concentration, buffer composition conditions, and the like are different, it is difficult to optimize these conditions.

  In addition, the microinjection method can introduce a gene with a very high probability only to a specific desired cell, and introduces a gene into the nucleus and cytoplasm separately, and the size of the gene that can be introduced is particularly limited. There are advantages such as not being. However, special equipment and techniques are required to introduce genes by this method. In particular, since a gene is introduced by inserting a fine glass tube into each very small cell, a very advanced technique is required, and even a skilled person is limited to the number of cells that can be introduced. There is.

  In addition, the viral vector method is limited in the size of the gene that can be introduced, and no matter how attenuated it is, it does not change the infection of the virus that is the pathogen, and it will cause defects in the target cells. Is also a concern.

  In addition, in the gene introduction device described in Patent Document 1, although the idea of incorporating a negatively charged gene into a cell by applying a voltage is excellent, the cytoplasm present in the cell, , There is a drawback of overlooking the presence of intracellular fluid formed in the presence of various ions.

  Although it can be easily imagined by considering an example of electrophoresis that is often used as a gene fractionation method, in general, the amount of movement of a substance (ion) by application of a voltage is inversely proportional to the molecular weight.

  For example, the aforementioned intracellular fluid contains a large amount of sodium ions and potassium ions as cations having a small molecular weight. If a voltage from the electrode (cathode) is applied under these conditions, sodium ions and potassium ions with lower molecular weight will move preferentially over genes with higher molecular weight, and the applied electricity It is not possible to use this power efficiently to move only the target gene. Furthermore, on the anode side, for example, chlorine ions, which are anions present in the cell, may be electrolyzed to generate chlorine gas.

  The present invention provides a new gene transfer apparatus and gene transfer method in addition to the gene transfer apparatus and the gene transfer method described above, and transforms the target cells with simple operations reliably and in large quantities. Alternatively, the problem is to introduce a gene prepared for transduction or the like.

  As will be described in detail in the following embodiments, a gene introduction apparatus for introducing a gene into a target cell, the first electrode connected to the cathode of a power source, and disposed in front of the first electrode A first buffer solution holding unit for holding a buffer solution, a first cation exchange membrane disposed further on the front side of the first buffer solution holding unit, a gene disposed on the further front side of the first cation exchange membrane, The gene transfer apparatus is configured to have at least a gene holding part that holds the gene and a first anion exchange membrane disposed in front of the gene holding part.

  As a result, only the gene intended for introduction can be reliably introduced into the cell, and the efficiency of gene introduction does not decrease due to the release of counter ions (cations) from within the cell.

  Further, a needle may be disposed on the front surface of the first anion exchange membrane.

  Thereby, even if it is a plant cell which has a cell wall in the outer periphery of a cell membrane, it becomes possible to introduce | transduce a gene very efficiently.

  Also, a second electrode connected to the anode of the power source, a second buffer solution holding part that is arranged in front of the second electrode and holds a buffer solution, and arranged further in front of the second buffer solution holding part Second anion exchange membrane, an electrolyte solution holding part that is further disposed on the front surface of the second anion exchange membrane and that holds the electrolyte solution, and a second cation exchange that is further disposed on the front surface of the electrolyte solution holding part The gene introduction device may be configured to have at least a membrane.

  Thereby, it can prevent that anions move to the electrode side of the counter electrode of the electrode which is going to introduce | transduce a gene, and generate | occur | produce gas, such as chlorine gas, for example.

  Furthermore, a needle may be disposed on the front surface of the second cation exchange membrane.

  Thereby, even if it is a plant cell which has a cell wall in the outer periphery of a cell membrane, it becomes possible to introduce | transduce a gene very efficiently.

  Moreover, you may comprise the cation of the electrolyte solution hold | maintained at an electrolyte solution holding part with the substance which can be metabolized by the metabolic pathway which exists in a target cell.

  Thus, the cation introduced together with the gene is treated under optimal conditions by the homeostasis inherent to the cell itself, and the influence on the cell can be suppressed.

  Moreover, you may comprise with the microscope which can observe the contact state of a said 1st anion exchange membrane and a target cell.

  Accordingly, since the operation can be performed while confirming the contact state between the first anion exchange membrane through which the gene passes and the target cell, the gene can be introduced more efficiently.

  Moreover, you may comprise with the monitor apparatus which can project the observation image of the said microscope.

  This frees the user from looking through the microscope and has the advantage that the operation can be easily performed.

  Further, as will be described in the following embodiments, a gene introduction apparatus for introducing a gene into a target cell, which is disposed on the front surface of the first electrode connected to the anode of the power source. A first buffer solution holding unit for holding a buffer solution, a first anion exchange membrane disposed further in front of the first buffer solution holding unit, and a gene further disposed in front of the first anion exchange membrane. The gene introduction device may be configured to have at least a gene holding unit that holds the first cation exchange membrane disposed in front of the gene holding unit.

  Thereby, for example, even when a gene that is originally charged with a negative charge is configured to be charged with a positive charge as a whole by, for example, chemical modification, gene transfer can be performed. For this, the gene itself may be modified, or the capsule itself may be modified by incorporating the gene into a microcapsule such as a lipid.

  Further, as will be described in detail in the following embodiments, a gene introduction method for introducing a gene into a target cell, wherein the gene is introduced via an ion exchange membrane directly in contact with the cell membrane of the target cell. It is to be introduced.

  As a result, it is possible to prevent a decrease in gene transfer efficiency caused by a cation having a molecular weight smaller than that of the gene to be transferred moving from the inside of the cell.

  Further, as will be described in detail in the following embodiment, a gene introduction method for introducing a gene into a target cell, wherein the gene is introduced via an ion exchange membrane directly in contact with the cell wall of the target cell. It is to be introduced.

  As a result, it is possible to prevent a decrease in gene transfer efficiency caused by a cation having a molecular weight smaller than that of the gene to be transferred moving from the inside of the cell. In addition, genes can be introduced without the need to remove cell walls.

  A gene prepared for transformation or transduction can be introduced into a target cell reliably and in a large amount with a simple operation.

  Hereinafter, an example of an embodiment of the present invention will be described in detail with reference to the accompanying drawings.

  FIG. 1 is a conceptual configuration diagram conceptually showing a gene introduction apparatus 100 according to the present invention.

  The gene introduction device 100 includes a lower plate (non-working side electrode structure) 120 that serves as a dish for target cells into which a gene is to be introduced, and a lower support 104 that supports the lower plate. On the upper side of the lower plate 120, an upper plate (working side electrode structure) 140 in which a gene to be introduced is enclosed and an upper support 106 that supports the upper plate are disposed to face each other. The upper support 106 is provided with a height adjustment mechanism 108 that can adjust the height of the upper plate 140. In FIG. 1, the height adjusting mechanism 108 is not necessarily shown in detail, but a mechanism capable of reliably adjusting the height of the upper plate 140 (distance from the lower plate 120) in units of micrometers. It has become. The adjustment of the adjustment mechanism is adjusted by the controller 152, for example.

  A microscope 102 that can observe the contact state between the two plates (more precisely, the contact state between the upper plate 140 and the target cells) is disposed near the contact surface between the lower plate 120 and the upper plate 140. Also with respect to this microscope, for example, a photographed image that is operated by the controller 152 can be displayed on the monitor 150.

  The power source 112 is a power source capable of generating a voltage applied to introduce a gene, and has a cathode connected to the upper plate side 140 side and an anode connected to the lower plate 120 side. .

  A power source for driving the height adjusting mechanism 108, the microscope 102, the controller 152, the monitor 150, and the like of the gene introduction apparatus 100 is separately provided.

  Further, the lower plate 120 and the upper plate 140 described above are designed to be removable.

  Next, the configurations of the lower plate 120 and the upper plate 140 will be described in more detail.

  FIG. 2 is a cross-sectional view showing a state where the lower plate 120 and the upper plate 140 are in contact with each other via the target cell 160.

  First, the upper plate 140 side will be described.

  The upper plate 140 includes a first electrode 142 connected to a cathode of a power source inside an upper plate case 141 functioning as a plate container. Furthermore, a first buffer solution holding part 143 that holds a buffer solution is disposed on the front surface of the first electrode 142. Furthermore, a first cation exchange membrane 144 is disposed on the front surface of the first buffer solution holding unit 143. Furthermore, a gene holding part 145 for holding a gene is provided in front of the first cation exchange membrane 144. Further, a first anion exchange membrane 146 is disposed on the front surface of the gene holding unit 145. The first anion exchange membrane 146 can directly contact the cell membrane or cell wall of the target cell 160.

  Next, the configuration of the lower plate 120 will be described in detail.

  The lower plate 120 includes a lower plate case 121 that functions as a container for the lower plate, and a second electrode 122 that is connected to the anode of the power source inside the lower plate case 121. Furthermore, a second buffer solution holding unit 123 that holds a buffer solution is disposed on the front surface of the second electrode 122. Further, a second anion exchange membrane 124 is disposed on the front surface of the second buffer solution holding unit 123. Further, an electrolyte solution holding part 125 for holding the electrolyte solution is disposed on the front surface of the second anion exchange membrane 124. Further, a second cation exchange membrane 126 is arranged on the front surface of the electrolyte solution holding unit 125. The second cation exchange membrane 126 is configured to be able to directly contact the cell membrane or cell wall of the target cell 160.

  The “front surface” here means the side closer to the target cell into which the gene is to be introduced.

  The lower plate case 121 and the upper plate case 141 are provided in the buffer solution holding units 123 and 143, the electrolyte solution holding unit 125, the gene holding unit 145 and the like without leaking. Consists of non-reactive (inert) material.

  Any conductive material can be used for the first electrode 142 and the second electrode 122, but in the case where each of the buffer solution holding portions 123 and 143 described later is present as in the present embodiment, the carbon electrode It is preferable to use (carbon electrode).

  Next, each buffer holding part 123, 143 holds a buffer solution for ensuring the electrical conductivity of the gene transfer device for a long time. Examples of the buffer solution include phosphate buffered saline and organic acids. It is possible to use an aqueous solution. More preferably, the electrolyte is more easily oxidized or reduced than the electrolytic reaction of water (oxidation reaction at the anode electrode and reduction reaction at the cathode electrode), for example, inorganic compounds such as ferrous phosphate and ferric phosphate, ascorbic acid By using substances such as (vitamin C) and sodium ascorbate, organic acids of phosphoric acid, oxalic acid, malic acid, succinic acid, fumaric acid and / or their salts, or mixtures thereof, gas from electrolysis of water It is also possible to prevent the occurrence of this and the increase in the conductive resistance or the fluctuation of the pH value due to this. Of course, it is not intended to be limited to the substances described here.

  Next, the cation exchange membranes 126 and 144 are ion exchange membranes having a function of selectively permeating cations. For example, Neocepta CM-1, CM-2, CMX, CMS, manufactured by Tokuyama Corporation. A cation exchange membrane such as CMB can be used without any particular limitation. Also, a cation exchange membrane of a type in which a cation exchange resin is filled in part or all of the pores of a porous film made of polyolefin resin, vinyl chloride resin, fluorine resin, polyamide resin, polyimide resin, etc. is particularly preferably used. can do. In this case, the cation exchange resin is filled, for example, by impregnating the pores of the porous film with a solution in which a crosslinking initiator such as styrene-divinylbenzene or chloromethylstyrene-divinylbenzene is mixed. Polymerization can be performed later, and a cation exchange group such as a sulfonic acid group, a carboxylic acid group, or a phosphonic acid group is introduced into the polymer.

  Next, the anion exchange membranes 124 and 146 are ion exchange membranes having a function of selectively permeating anions. For example, Neocepta AM-1, AM-3, AMX, AHA manufactured by Tokuyama Corporation. Anion exchange membranes such as AMH and ACS can be used without particular limitation. Further, an anion exchange membrane of a type in which an anion exchange resin is polymerized in part or all of the pores of a porous film made of a polyolefin resin, a vinyl chloride resin, a fluorine resin, a polyamide resin, or a polyimide resin is particularly preferably used. be able to. In this case, the anion exchange resin is filled after impregnating the pores of the porous film with a solution in which a polymerization initiator is blended with a crosslinking monomer such as styrene-divinylbenzene or chloromethylstyrene-divinylbenzene. The polymerization can be carried out by introducing an anion exchange group such as a primary to tertiary amino group, quaternary ammonium group, pyridyl group, imidazole group, quaternary pyridinium group, or quaternary imidazolium group into the polymer.

  Next, the gene holding unit 145 holds a gene to be introduced into the target cell 160. The gene holding unit 145 may hold the gene as a solution, for example, or may be provided in a form such as a polyacrylamide gel or an agarose gel.

  In addition, “gene” in the present specification includes not only DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), but also DNA-RNA hybrids. Furthermore, not only those present as double strands but also single strands are included, and further, those having a circular structure such as a plasmid are also included.

  Next, the electrolyte solution holding unit 125 holds an electrolyte solution that is a substance that can be metabolized by a metabolic pathway in which the cation originally exists in the target cell 160. For example, when potassium ion or sodium ion is present as a cation, the cation is introduced into the target cell 160 by energization of the gene introduction device 100, but is present on the cell membrane of the target cell 160. The concentration is appropriately adjusted by a sodium pump or a potassium pump. Specifically, physiological saline or the like can be used.

  Next, a gene introduction method using the gene introduction apparatus 100 will be described.

  First, it is necessary to appropriately adjust the target cell 160 to which the gene is to be introduced and prepare it on the upper surface of the lower plate 120, that is, the upper surface of the second cation exchange membrane 126.

  Subsequently, the upper plate 140 is moved to the lower plate 120 side by the height adjusting mechanism 108. At this time, adjustment is performed while observing the monitor 150 on which the image of the microscope 102 is projected so as not to destroy the target cell 160 by pressing the upper plate 140 too far to the lower plate 120 side. However, this adjustment work is shown as a desirable work and is not an essential work.

  By this adjustment, the target cells 160 are preferably arranged so as to contact both the second cation exchange membrane 126 in the lower plate 120 and the first anion exchange membrane 146 in the upper plate 140. In FIG. 2, the target cells 160 are neatly arranged in a line (in one layer). However, even if the target cells 160 overlap each other, the effect of the present invention is not hindered.

  In this state, by operating the controller 152, a voltage is applied from the power source 112 to the upper plate 140 and the lower plate 120. When the voltage from the power source 112 is transmitted to the electrodes 122 and 142, the following operation occurs.

  First, the following action occurs in the lower plate 120.

  When a current is transmitted from the power source 112 to the second electrode 122, the buffer solution provided in the second buffer solution holding unit 123 is oxidized. For example, in the case of ferrous phosphate, it changes to ferric phosphate. If it does so, the balance of the cation and anion with which this 2nd buffer solution holding part 123 is equipped will be broken (a cation will increase). In order to compensate for this balance, the cations in the second buffer solution holding part 123 move in the direction of the electrolyte solution holding part 125 and try to keep the balance. On the other hand, the anion provided in the electrolyte solution holding unit 125 also moves to the second buffer solution holding unit 123 side and tries to maintain the balance. However, due to the presence of the second anion exchange membrane 124 positioned between the second buffer solution holding unit 123 and the electrolyte solution holding unit 125, cations cannot pass through, and only anions pass selectively. That is, the movement of cations from the second buffer solution holding unit 123 to the electrolyte solution holding unit 125 is not recognized, and only the movement of anions from the electrolyte solution holding unit 125 to the second buffer solution holding unit 123 is allowed. Then, this time, the balance between the cation and the anion of the electrolyte solution holding part 125 is lost. Furthermore, in order to compensate for this loss of balance, the cations in the electrolyte solution holding unit 125 pass through the second cation exchange membrane 126 and move to the outside (target cell side) of the lower plate 120 to maintain the balance. On the other hand, from the inside (cytoplasm) of the target cell 160 in contact with the second cation exchange membrane 126, in order to compensate for this loss of balance, the anion in the cytoplasm moves to the electrolyte solution holding unit 125 to maintain the balance. To do. However, due to the presence of the second cation exchange membrane 126 located between the electrolyte solution holding unit 125 and the target cell 160, the anion cannot pass, and only the cation passes selectively. That is, the movement of the anion from the cytoplasm of the target cell 160 to the electrolytic solution holding part 125 is not recognized, and only the movement of the cation from the electrolytic solution holding part 125 to the target cell 160 side is allowed. Since the cations at this time are selectively permeated through the second cation exchange membrane 126, they can move to the target cell 160 side.

  Next, the operation inside the upper plate 140 will be described.

  When current is transmitted from the power supply 112 to the first electrode 142, the buffer solution provided in the first buffer solution holding unit 143 is reduced. For example, in the case of ferric phosphate, it changes to ferrous phosphate. If it does so, the balance of the cation and anion with which the 1st buffer solution holding part 143 is equipped will be broken (anion will increase). In order to compensate for this balance, the anion of the first buffer solution holding unit 143 tends to move in the direction of the gene holding unit 145. On the other hand, the cations provided in the gene holding unit 145 also move in the direction of the first buffer holding unit 143 and try to maintain the balance. However, due to the presence of the first cation exchange membrane 144 located between the first buffer holding unit 143 and the gene holding unit 145, the anion cannot pass through and only the cation is selectively permeated. That is, the movement of the anion from the first buffer holding unit 143 to the gene holding unit 145 is not recognized, and only the movement of the cation from the gene holding unit 145 to the first buffer holding unit 143 is allowed. In this case, the balance between the cation and anion of the gene holding unit 145 is lost. Furthermore, in order to compensate for this loss of balance, the anion of the gene holding part 145 moves through the second anion exchange membrane 146 to the outside of the upper plate 140 (target cell side) and tries to maintain the balance. On the other hand, from the inside (cytoplasm) of the target cell 160 in contact with the second anion exchange membrane 146, in order to compensate for this loss of balance, the cations in the cytoplasm move to the gene holding unit 145 and try to maintain the balance. . However, due to the presence of the second anion exchange membrane 146 located between the gene holding unit 145 and the target cell 160, the cation cannot pass through and only the anion passes through selectively. That is, the movement of the cation from the cytoplasm of the target cell 160 to the gene holding unit 145 is not recognized, and only the movement of the anion from the gene holding unit 145 to the target cell 160 side is allowed. Since the anion (gene) at this time is selectively permeated through the first anion exchange membrane 146, it can move to the target cell 160 side.

  Through the action in the lower plate 120 and the upper plate 140, the gene provided in the upper plate 140 moves into the target cell 160. Contrary to the above description, when the current from the power source 112 is de-energized, gene introduction is immediately stopped. That is, by appropriately controlling the energization / non-energization from the power source 112, the amount of gene to be introduced can be appropriately controlled.

  In addition, as shown in FIG. 3, a gene from the gene holding unit 145 is introduced into the target cell 160 through the first anion exchange membrane 146 and at the same time, cations provided in the electrolyte holding unit 125 are present. An equivalent amount of ions is introduced through the second cation exchange membrane 126. Thus, since the total amount of cations and anions introduced is introduced while being relatively controlled, the pH value in the cytoplasm of the target cell 160 does not change significantly. Furthermore, by configuring the cation provided in the electrolyte solution holding unit 125 with, for example, NAD + (nicotinamide adenine dinucleotide phosphate) or the like, a metabolic pathway (for example, a citrate circuit or the like originally present in the target cell 160). Since it is consumed by being used appropriately in an electron transfer system or the like, there is no problem in the target cell 160 due to accumulation of introduced cations.

  Next, another embodiment of the gene introduction apparatus will be described.

  FIG. 4 shows another embodiment of the present invention and corresponds to FIG. 3 in the gene introduction apparatus 100 described above.

  The feature of this embodiment is effective when applied when the target cell 260 is a plant cell. Unlike animal cells, plant cells have a cell wall 260H made of cellulose or the like on the outer side of the cell membrane 260M. By applying the present invention, it is possible to introduce a gene without being affected by the presence of the cell wall 260H by using the gene introduction device 100 described above. However, as shown in FIG. 4, on the surface of the first anion exchange membrane 246 and the second cation exchange membrane 226 that are in direct contact with the cell wall 260H of the plant target cell 260, a plurality of needles (hollow) made on the nano level are provided. And further desirable), the gene to be introduced partially penetrates the cell wall 260H of the plant target cell 260, and moves through the gap that penetrates the plant target cell 260 into the plant target cell 260. Thus, the gene introduction efficiency of the present invention can be further increased by providing an infinite number of needles.

  Furthermore, in the present invention, it is not always necessary to prepare target cells for gene transfer at the cell level. As shown in FIG. 5, the gene is directly introduced into an organism (the leaf of a plant 190 in the figure). It is also possible to do. As shown in FIG. 5, a structure (working electrode structure) corresponding to the upper plate on which the gene to be introduced is held on the front and back surfaces of the leaves of the plant 190, and a lower plate on the opposite side It can be introduced by arranging a structure (non-working side electrode structure) to be applied and applying a voltage from a power source. At this time, since a cell wall made of cellulose, a cuticle layer, and the like are formed on the surface of the plant body, the device may be used after removing these layers with a chemical or the like.

  Although not shown, the gene holding part can be provided on the structure side connected to the anode. When a gene that is originally charged with a negative charge is configured to be charged with a positive charge as a whole by, for example, chemical modification, gene transfer is possible even with such a configuration. It becomes. For this, the gene itself may be modified, or the capsule itself may be modified by incorporating the gene into a microcapsule such as a lipid. Furthermore, by adopting such a configuration, both of which function as “non-acting side structures” into which genes are not introduced can both function as working side structures.

  In the present specification, the term “acting side” in the terms of a working side structure and a non-acting side structure means a side on which a gene holding action occurs and a gene introduction action occurs.

  The present invention can be applied as a gene introduction apparatus for gene introduction widely regardless of animal cells or plant cells.

The conceptual block diagram which comprised notionally the gene introduction apparatus 100 which is an example of embodiment of this invention Cross-sectional configuration diagram of the upper plate and the lower plate in FIG. The figure which showed the movement of the anion and cation in a target cell at the time of application of the gene introduction apparatus 100 Example of arrangement with innumerable needles on the contact surface between the ion exchange membrane and target cells Conceptual diagram for direct gene transfer into a plant Gene transfer device described in Patent Document 1

Explanation of symbols

DESCRIPTION OF SYMBOLS 100 ... Gene transfer apparatus 102 ... Microscope 104 ... Lower support body 106 ... Upper support body 108 ... Height adjustment mechanism 112 ... Power supply 120 ... Lower plate 121 ... Lower plate case 122 ... 2nd electrode 123 ... 2nd buffer solution holding part 124 ... second anion exchange membrane 125 ... electrolyte holding part 126 ... second cation exchange membrane 140 ... upper plate 141 ... upper plate case 142 ... first electrode 143 ... first buffer holding part 144 ... first cation exchange membrane 145 ... Gene holding unit 146 ... first anion exchange membrane 150 ... monitor 152 ... controller 160 ... target cell 160C ... cell nucleus 160M ... cell membrane 190 ... plant 270 ... needle

Claims (10)

A gene introduction device for introducing a gene into a target cell,
A first electrode connected to the cathode of the power source;
A first buffer solution holding unit disposed on the front surface of the first electrode and holding a buffer solution;
A first cation exchange membrane disposed further on the front surface of the first buffer holding unit;
A gene holding part that is arranged in front of the first cation exchange membrane and holds a gene;
A first anion exchange membrane disposed further in front of the gene holding unit;
A gene introduction apparatus comprising at least In claim 1,
A gene introduction device, wherein a needle is disposed on a front surface of the first anion exchange membrane. In claim 1 or 2,
A second electrode connected to the anode of the power source;
A second buffer solution holding unit disposed on the front surface of the second electrode and holding a buffer solution;
A second anion exchange membrane disposed further in front of the second buffer holding part;
An electrolyte solution holding part that is disposed further on the front surface of the second anion exchange membrane and holds the electrolyte solution;
A second cation exchange membrane disposed further in front of the electrolyte solution holding unit;
A gene introduction apparatus comprising at least In claim 3, further:
A gene introduction device, wherein a needle is disposed on a front surface of the second cation exchange membrane. In claim 3 or 4,
The gene introduction device, wherein the cation of the electrolyte solution held in the electrolyte solution holding part is a substance that can be metabolized by a metabolic pathway existing in the target cell. In any one of Claims 1 thru | or 5, Furthermore,
A gene introduction apparatus comprising a microscope capable of observing a contact state between the first anion exchange membrane and a target cell. In claim 6, further:
A gene introduction apparatus comprising a monitor device capable of projecting an observation image of the microscope. A gene introduction device for introducing a gene into a target cell,
A first electrode connected to the anode of the power source;
A first buffer solution holding unit disposed on the front surface of the first electrode and holding a buffer solution;
A first anion exchange membrane disposed further in front of the first buffer holding part;
A gene holding part that is arranged in front of the first anion exchange membrane and holds a gene;
A first cation exchange membrane disposed further in front of the gene holding unit;
A gene introduction apparatus comprising at least A gene introduction method for introducing a gene into a target cell,
A gene introduction method comprising introducing a gene through an ion exchange membrane directly in contact with a cell membrane of a target cell. A gene introduction method for introducing a gene into a target cell,
A gene introduction method comprising introducing a gene through an ion exchange membrane directly in contact with a cell wall of a target cell.

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