JPH0678742A - Substance introducing system - Google Patents

Abstract

PURPOSE:To provide the system enabling substance to be introduced into a specimen, simplified in vacuum operation as well as structure by executing vacuum control for a single vacuum chamber. CONSTITUTION:The system, that is a gene introducing system, is made up of (A) a vessel 1 so designed that the front face serves as a closure member 11, an opening 140 is provided at the top face 14, a specimen is located at the lower part where is also provided with a vacuum chamber, (B) a mechanism 2 fixed firmly to the top face of the vessel 1 and to emit nylon projectiles provided with gene-carrying particles through the opening 140, (C) members 30 stuck to the left and right inner walls of the vessel 1, i.e. a stop plate supporting device 3 with a support 31 located at the center, (D) a stop plate 4 located at the support 31, to receive nylon projectiles emitted from the mechanism 2 and make gene-carrying particles fly via a hole 41, to the specimen T, (E) a dustproof cover 5 to block tailings, etc., from the side and upper left and right of the specimen, and (F) a mesh member 6 placed on the top end of the dustproof cover 5 and to trap thready chips.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention is directed to hitting a stop plate supported by a stop plate supporting device in a vacuum chamber with a bullet piece so that a gene or other introduced substance carried on the bullet piece is a living cell or tissue. The present invention relates to a substance introduction device to be introduced into other samples.

[0002]

2. Description of the Related Art Conventionally, as a material introduction apparatus of this type disclosed in Japanese Patent Laid-Open No. 1-227764, a vacuum chamber C formed in a housing means H has upper and lower chambers with a horizontal portion M as shown in FIG. Divided into UC and DC,
Upper and lower chambers UC and DC are surge tanks S
Shut-off valves UV, DV and filters UF, DF communicating with
Independent chambers that are independently vacuum-controlled by the conduits UL and DL having The collision receiving mechanism R is arranged at the opening of the horizontal portion M, and the multi-shot piece ejected by the propulsion means P should collide with the impact stop member R and be discharged from the central opening O into the microorganism sample BS. In some cases, a small bullet covered with a substance is sent into a biological material as a sample, and a multi-bullet closes the central opening O and seals the upper and lower chambers UC and DC.

[0003]

In the above-mentioned conventional substance introducing apparatus, the inside of the vacuum chamber C is divided into the upper and lower independent vacuum chambers UC and DU by the horizontal portion M. Therefore, the upper and lower vacuum chambers UD and Since the DC vacuum control needs to be independently performed by the shutoff valves UV and DV and the conduits UL and DL, the vacuum control operation is difficult and the structure is complicated.

Therefore, the inventors of the present invention removed the horizontal member for partitioning the vacuum chamber into upper and lower parts to form one vacuum chamber, and used a stop plate supporting mechanism of a small area as a stop plate for stopping the bullets in the vacuum chamber. Supported by, and paying attention to the technical idea of arranging the sample facing the stop plate, filter and shield dust and other things caused by collision of ammunition piece with the stop plate and explosion of explosive to the sample. As a result of further research and development focusing on the technical idea of not reaching the target, the substance introduction device enables the introduction of the substance into the sample and the vacuum control by controlling the vacuum of one vacuum chamber. The present invention has been achieved which achieves the objective of simplifying the operation and simplifying the structure.

[0005]

The substance introduction apparatus of the present invention (the first invention according to claim 1) is a container which forms a vacuum chamber, has an opening, and which holds a sample into which a substance is introduced. A firing mechanism that fires a piece of bullet with an introduced substance through an opening, a stop plate support device that is fixed to a container and has support portions arranged at intervals with respect to a sample placed in a vacuum chamber, and a stop plate A member arranged in the support portion of the support device and having a hole in the center, which receives a piece of bullet fired from the firing mechanism and at the same time causes the introduced substance to fly toward the sample through the hole. It consists of and.

The substance introduction apparatus of the present invention (the second invention according to claim 2) is a container which forms a vacuum chamber, has an opening, and in which a sample into which a gene is introduced is placed, and a gene is loaded from the opening. A firing mechanism for firing a bullet provided with particles, a stop plate support device in which a support portion is arranged at a distance from a sample fixed in a container and placed in a vacuum chamber, and a stop plate support device A stop plate which is arranged in the support part and has a hole in the center, which receives a piece of bullet fired from a firing mechanism and at the same time causes particles carrying a gene to fly toward the sample through the hole. It consists of and.

The substance introducing apparatus of the present invention (the third invention according to claim 3) is arranged between the sample placed on the container and the stop plate, and passes through the hole of the stop plate as compared with the first invention. A filter device that filters incoming thread-like debris and debris so that it does not reach the sample, and parts other than the filter device are shielded so that debris does not reach the sample without going through the filter device. A shielding member is added.

[0008]

The substance introducing apparatus of the first invention having the above-mentioned structure is
A projecting piece is fired by the firing mechanism, and the projecting piece collides with the stop plate arranged in the vacuum chamber by the supporting portion of the stop plate supporting device and is received, and the introduced substance is passed through the hole. The material is introduced into the sample by flying toward the sample.

In the substance introducing apparatus of the second aspect of the present invention having the above-mentioned structure, the projecting mechanism ejects the bullet having the particles carrying the gene, and the bullet is placed in the vacuum chamber by the supporting portion of the stop plate supporting device. In addition to colliding with the stop plate and being received, particles carrying the gene are caused to fly toward the sample through the hole to introduce the gene into the sample.

In the substance introducing device according to the third aspect of the present invention having the above structure, the projecting mechanism ejects a piece of the bullet containing the introduced substance,
The projectile collides with the stop plate arranged in the vacuum chamber and is received by the supporting portion of the stop plate supporting device.
The introduced substance is made to fly toward the sample through the hole to introduce the substance into the sample, and at the same time, the thread-like debris and dust passing through the hole of the stop plate are filtered by the filter device and the filter device. The dust that reaches the sample through a path that does not include is blocked by the blocking member to prevent it from reaching the sample.

[0011]

The substance introducing apparatus of the first invention having the above-described action has the effect of being able to introduce a substance into a sample, and has only one vacuum chamber in the container. Easy to operate vacuum control,
Moreover, there is an effect that the structure is simple.

The substance introduction device of the second invention having the above-described action has the effect of being able to introduce a gene into a sample and has the same effect as the first invention.

The substance introducing device of the third invention having the above-mentioned action exhibits the effect of the first invention, and at the same time, removes the dust passing through the hole of the stop plate, the thread-like fragments and the dust reaching at other times. Since it is blocked by the filter device and the shielding member, it is possible to introduce only the necessary substance into the sample.

[0014]

Embodiments of the present invention will now be specifically described with reference to the drawings.

(First Example) As an example, the substance introduction apparatus of the first example uses a plasmid in which 35S / GUS gene is incorporated into a slice of a young leaf of corn, which is the second leaf after one week of germination of sample T. It is loaded with tungsten particles and shot in.

The substance introducing apparatus of the first embodiment is the embodiment of the first to third inventions, and as shown in FIG. 1, a vacuum chamber 10 is formed, an opening 14O is formed in an upper surface portion 14 and a lower portion is formed. A container 1 in which a sample is placed and an opening 13 communicating with the vacuum suction pump 12 is formed on the other wall surface, and a nylon bullet provided with particles carrying a gene as an introduced substance through an opening 14O fixed to the upper surface of the container 1 And a member 30 fixed to the left and right inner walls of the container 1 and having a support portion 31 arranged at the center, and a stop plate supporting device 3 arranged at the support portion 31 of the stop plate supporting device 3. The stop plate 4 that receives the nylon bullets fired from the firing mechanism 2 and causes the particles carrying the gene, which is the introduced substance, to fly to the lower sample T through the hole 41, and the lateral side of the sample. And a dust-proof cover 5 that constitutes a shielding member that shields dust and the like from diagonally above, and a filter device that filters dust and thread-like debris that is placed on the upper end of the dust-proof cover 5 and that passes through the hole 41 of the stop plate 4. And a mesh member 6 constituting the.

The container 1 is composed of a hollow rectangular parallelepiped member 10 and a lid member 11 whose front portion is openable and closable. It is opened when a sample or nylon bullet is put in or taken out, and is otherwise airtight. It consists of a closed structure. A sample shelf 15 on which the sample T is placed is arranged at a position slightly apart from the bottom of the lower portion of the container 1. A container 16 having a trapezoidal cross section having an opening 13 formed in an approximately center of a side wall on the right side in the drawing and having an inclined surface on the front face arranged adjacently
A pipe 12P provided with a valve 12V by a suction pump 12 driven by an electric motor 12M arranged inside
The inside of the container 1 is sucked through the via to maintain the inside of the vacuum chamfer 10 in a vacuum state.

The firing mechanism 2 includes a vacuum cylinder 21 fixed to the upper surface of the container 1, a barrel 22 coaxially arranged in the vacuum cylinder 21, and an anesthesia gun coaxially arranged above the barrel 22. It is composed of a gene gun 23 composed of an equivalent special launching device. The gene gun 23 is filled with tungsten or gold particles having a diameter of 1 to 5 microns at its head, and a nylon bullet made of nylon having a diameter of 5.6 mm and gunpowder are loaded.
Nylon bullets pass through the barrel 22 due to the explosion of gunpowder,
It is configured to inject into the container 1 through the opening 14O on the upper surface of the container 1.

The stop plate supporting device 3 is composed of a substantially I-shaped member having an area of about one-third of the cross-sectional area of the vacuum chamber 10, and is located at a position about one-third above the container 1. Is horizontally fixed to the inner wall of the container 1 at the left and right ends, and a support portion 31 for fixing the stop plate 4 to the center of a rectangular portion connecting the left and right ends is coaxial with the barrel 22 of the firing mechanism 2. It is installed in.

The stop plate 4 is a support portion 3 of the stop plate support device 3.
A hole 41 having a diameter of 1 mm is coaxially formed in the center, which is composed of an annular plate 40 fixed to 1.

The dust-proof cover 5 comprises a hollow cylindrical body 50 having an inner diameter larger than the sample and a height several times higher than the sample and having openings 51 and 52 formed at both ends. The lower end opening 52 is coaxial with the sample on the sample shelf 15. Arrange.

The mesh member 6 is composed of a mesh of a size that captures dust and thread-like debris passing through the hole 41 of the stop plate 4 and allows only particles carrying the gene as the introduced substance to pass through. It is a circular member and is placed on the upper end of the dustproof cover 5 so as to cover the opening 51.

In the apparatus of the first embodiment having the above structure, the gene gun 23 of the firing mechanism 2 turns 35S into tungsten particles.
A nylon bullet carrying a plasmid incorporating the / GUS gene passes through the barrel 22 by the explosion of gunpowder and is fired to collide with the stop plate 4 supported by the stop plate support device 3. The stop plate 4 allows particles carrying a gene as an introduced substance to be maintained in a vacuum state in a vacuum chamber through a hole 41 and fly through a space with little resistance toward a sample T which is a young leaf of corn, and a mesh member. The substance passes through 6 and reaches the sample T, and some of the carrier fine particles rupture the cell wall together with the substance fragment and invade into the cell, stop there, and release the substance fragment, whereby the substance is introduced into the sample T. .

Of the dust generated when the nylon bullet collides with the stop plate 4 and the dust and thread-like debris that have passed through the hole 41 of the stop plate 4, the sample T is put on the strong gas flow generated by the combustion of the explosive. Those flying from the side and diagonally above are blocked by the dust-proof cover 5, and those flying from above the sample T are blocked by the mesh member 6.

The device of the first embodiment having the above-mentioned operation is the GU
The S substance can be introduced into the cells of the sample T in an appropriate state.

In the apparatus of the first embodiment, the dust cover 5 and the mesh member 6 ride on the strong flow of the gas generated by the combustion of the explosive and collide with the stop plate 4 of the nylon ammunition, and other dust and thread-like particles are generated. It has an effect of preventing the debris from reaching the sample T, and an effect of preventing the cells of the sample T from being damaged, sometimes destroyed or killed.

Further, the space in which the sample T surrounded by the dust-proof cover 5 and the mesh member 6 is placed has an effect that the state in which it is not contaminated can be maintained.

(Second Embodiment) In the substance introducing apparatus of the second embodiment, the I-shaped stop plate supporting device 3 of the first embodiment has four arms as shown in FIG. A cross-shaped member 3 having a surface area of 1/10 or less of the cross-sectional area of the vacuum chamber
The number of arms is changed to 2, and four arms 32A whose both side surfaces are flat are arranged radially at 90-degree intervals.

When the nylon ammunition is fired between the four arms 32A, the arms 32A do not prevent the strong flow of the gas generated by the combustion of the explosive, and are uniformly radially downward along the flat surfaces thereof. Since the particles are guided so as to flow, there is no bias in the flow in the vacuum chamber 10 and there is no bias in the scattering of particles carrying the gene that is the introduced substance, and it is possible to reach the sample evenly and uniformly, and dusts and filamentous fragments Since the scattering of the particles is also uniform, there is an effect that the blocking efficiency of the dust cover 5 and the mesh member is high.

(Third Embodiment) In the substance introducing apparatus of the third embodiment, a dustproof screen is constituted by a square member 53 which abuts on the inner wall of the container 1, and the dustproof screen is provided between the stop plate supporting device 3 and the sample shelf 15. A circular hole portion 53H which is slightly larger than the sample T is formed in the central portion, and the mesh member 6 similar to that of the first embodiment is arranged in this hole portion 53H. The same effects as those of the first embodiment are achieved with only a slight difference.

(Fourth Embodiment) As shown in FIGS. 4 and 5, the material introducing device of the fourth embodiment has a stop plate supporting device 34 and a ring member 34R at the upper end on the upper wall of the container 1 having an opening 14O. The support portion 34S of the stop plate 4 is arranged in the center of the disc member 34C at the lower end, which is screwed onto and suspended by the three rod-shaped leg portions 34L.

In the fourth embodiment, the upper end of the container 1 has an upper wall 1
4, a hollow cylindrical dustproof screen 54 having an inner wall 54I having an inner diameter sufficiently larger than the outer diameter of the sample T is coaxially provided on the outer side of the stop plate supporting device 34, and the lower end opening is provided. The mesh member 6 similar to that of the first embodiment is fixed to the portion 54O.

The apparatus of the fourth embodiment having the above-mentioned configuration is the first
When a nylon bullet collides with the stop plate 4 as in the example, only the particles carrying the gene as the substance to be introduced have holes 4
1 through the mesh member 6 and the container 1
The substance is introduced into the sample T by reaching the sample T placed on the sample shelf.

The dust generated by the collision of the nylon bullets with the stop plate 4 and scattered laterally by the strong gas flow generated by the combustion of the explosive collides with the inner wall 54I of the dust-proof screen 54, The flight to the sample T is prevented.

The above-mentioned embodiments are given as examples for the purpose of explanation, and the present invention is not limited to them, and those skilled in the art can understand based on the claims, the detailed description of the invention and the description of the drawings. Modifications and additions can be made without departing from the technical idea of the present invention that can be recognized.

In the above-mentioned embodiment, the container is a rectangular parallelepiped, but the present invention is to make the flow in the container uniform,
From the viewpoint of eliminating the stagnation area, it is possible to employ a hollow cylindrical body or other container.

In the above embodiments, an example in which an annular plate is used as the stop plate has been described, but in the present invention, in order to increase the fixing area and obtain a strong fixing, the outer shape such as a square or a rectangle is rectangular. It is possible to employ a stop plate.

As an object of the sample of the present invention, an example of young leaves of corn was explained as an example in the above-mentioned embodiment, but there is no limitation in the target cell type, and from a huge cell type of the cell wall to a soft type, It is applicable not only to plant cells and certain types of microorganisms, but also to microorganisms without cell walls and animal cells.

The introduced substance is, for example, 35S / GUS.
Although the example of supporting the gene on the tungsten particles having the gene incorporated therein has been described in the examples, the introduced substance used in the present invention is not limited thereto, and other than that, the gene includes anthracene or a sedative which is a pigment. The genes involved in the production of useful substances such as scopolamine, the dwarfing genes that control the dwarfing of plants, the genes of the luciferase enzyme that controls firefly-derived luminescence, the BT insect gene and the coat of TMV. A gene that protects a plant, such as a gene encoding a protein, or a plasmid that incorporates these genes can be used.

In addition to tungsten particles, gold particles, ceramic particles, glass beads, latex particles and the like can be used as the particles carrying the gene.

As other introduced substances, capsules containing cytoplasm derived from other cells or physiologically active substances, particles for inspection / analysis such as magnetic fine particles and radioactive fine particles, and the like can be adopted.

[Brief description of drawings]

FIG. 1 is a partially cutaway perspective view showing a device according to a first embodiment of the present invention.

FIG. 2 is a partially cutaway perspective view showing a second embodiment device of the present invention.

FIG. 3 is a partially cutaway perspective view showing a device according to a third embodiment of the present invention.

FIG. 4 is an exploded perspective view showing a main part of a device according to a fourth embodiment of the present invention.

FIG. 5 is a vertical cross-sectional view showing a main part of a device according to a fourth embodiment of the present invention.

FIG. 6 is a perspective view showing a conventional device.

[Explanation of symbols]

 1 Container 2 Firing Mechanism 3 Stop Plate Support Device 4 Stop Plate 5 Dust Cover 6 Mesh Member 10 Vacuum Chamber 12 Vacuum Suction Pump 13, 14 Open 21 Vacuum Cylinder 22 Barrel 23 Gene Gun 31 Support 41 Hole

Claims (3)

[Claims] 1. A container which forms a vacuum chamber and has an opening, on which a sample for introducing a substance is placed, a firing mechanism for ejecting a piece of bullet containing the introduction substance through the opening, and a vacuum chamber fixed to the container. A stop plate supporting device in which a supporting part is arranged at a distance from the sample placed inside, and a member having a hole in the center, which is arranged in the supporting part of the stopping plate supporting device. A substance introduction device, comprising: a stop plate that receives a shot piece that has been ejected and causes the introduced substance to fly toward the sample through a hole. 2. The container according to claim 1, which forms a vacuum chamber, has an opening, and in which a sample into which a gene is introduced is placed, and a projecting mechanism for projecting a fragment equipped with particles carrying the gene from the opening. A stop plate supporting device in which a supporting part is arranged at an interval with respect to a sample fixed to a container and placed in a vacuum chamber; and a hole part in the center arranged in the supporting part of the stopping plate supporting device. A substance introduction device, which is a member, and which comprises a stop plate for receiving a piece of bullet fired from a firing mechanism and for causing the particle carrying the other gene to fly toward the sample through the hole. 3. The method according to claim 1, wherein thread-like debris and debris, which are disposed between the sample placed on the container and the stop plate and pass through the holes of the stop plate, are filtered so as not to reach the sample. And a shielding member that shields parts other than the filter device so that dust does not reach the sample without passing through the filter device.