KR100232556B1 - Particle bombarding device for transformation of cells - Google Patents

Abstract

The present invention is a tool used when mechanically inserting DNA (gene) into plant cells (usually called transformation). As the research and industry of genetic engineering develops, the demand for machines for this purpose also increases. There are several currently developed, but all have one or more disadvantages, so we have applied for a patent because we have created a new type of device that improves on this.

PDS-1000과 아직 상품생산은 되지 않으나 Particle Inflow Gun(PIG)이 잘 알려져 있고, 한국에서 생산되지만 널리 보급은 되지 않은 바이오니아(구, 한국생공)사의 Gene Gun IJ도 있다. 이들에서 나타나는 가장 큰 문제는 기계의 값이 사용빈도나 재료의 구성에 비하여 너무 비싸고 조작하는데 시간이 많이 걸리거나, 기능을 제대로 하지 못하는 것 등이있다.Existing representative machine is Bio-Rad's Biolistics

PDS-1000 and Particle Inflow Gun (PIG) are well known, but there is Gene Gun IJ of Bioneer (formerly Korea Biotechnology) produced in Korea but not widely distributed. The biggest problem in these cases is that the value of the machine is too expensive for the frequency of use or the composition of the material, it takes a long time to operate, or it does not function properly.

In the present invention, all the problems pointed out by the other device are solved. The problem was solved by simplifying the structure and working steps of the device and applying new methods. The key to the improvement was the first use of a separate piston and macroarrier, which prevented the propulsion gas from reaching the target by allowing the particle carrier to block the projection. Second, the gas receiving part of the solenoid applies the principle of an air valve, and the male part of the valve is used as a cylinder to simplify the attachment and detachment of particles. Third, unlike the above, when the particle injection kit is used, particles are directly projected under the gas pressure, so they can be used even at low gas pressures, and they are simple to use because they do not interfere with the drying step.

Description

Particle Projection Device for Cell Transformation

1 is a front view of the particle projection apparatus assembled.

2 is a front view showing the cylinder and the particle projection socket.

3 is a longitudinal section when the particle projection socket is assembled into a cylinder.

4 shows a particle spray kit and a longitudinal cross section when it is inserted into a particle spray socket.

1 is an overall configuration of the apparatus. One box (inside 15 × 15 × 25 cm) is used as the vacuum chamber 8 as a whole. The outlet of the solenoid 4 is connected to the outside of the solenoid 4 and the air valve socket 7 is directly connected to the inner surface through a hole having a diameter of 1.2 to 2.0 cm at the center of the upper surface of the box. The distance between them is as short as possible, and the inner diameter of the connection is not equal to or too large for the solenoid's orifice, which helps to maintain the inlet gas pressure. The order of the parts connected in the direction of gas inflow outside the box is the inlet of the solenoid-the gas pressure gauge of 150 kg (3)-the gas reservoir (creating a 30 ml empty space) (2)-the openable gas valve (1). The gas valve 2 is opened in the projection operation, passes the gas through the empty tube, and locks again after confirming that the pressure gauge has risen to the target value. The pressure here is consistent with the discharge pressure regulator (secondary meter) attached to the gas cylinder. The solenoid 4 uses a thing that opens and closes at a maximum of 207 kg / cm 2 (3000 PSI). It is connected to the timer 6 by the wire 5, and the timer is connected to the power switch again to control the opening and closing. The gas passage time of the timer is adjusted to 0.01 seconds.

The part connected with the air valve socket 7 in the vacuum chamber is, in turn, the cylinder 9-the particle carrier holder 10-the particle projection socket 11. The projected particles fall on the sample 15 (mainly plant cells) on the sample stage 16. The inside of the box is continued until the vacuum pump 20 is operated to form a vacuum of 25 Hg in the vacuum meter 19. If the inside of the vacuum chamber is still vacuum after the projection, open the valve of the air inlet 18 to inject air, and then open the door. The air inlet has a filter for disinfection. The effect of vacuum is to increase the scattering and cell penetration of the particles.

The gas ejected at high pressure blows the target sample (cell, tissue) placed on the sample stage, so that the pressure blocking plate 12 is tightly inserted under the particle projection socket to prevent this. At the center of the plate is a particle passage opening 13 which is connected to the particle projection hole of the particle projection socket. The plate is pushed through the grooves of the vacuum chamber wall after the particle projection socket is mounted. The high gas pressure in the upper part of the vacuum chamber after firing is distributed to the lower part through the pressure distribution hose (or tube) 14 connected through the wall, so that the air density of the entire vacuum chamber is quickly uniformed.

2 is an exploded view of the parts connected below the upper surface of the vacuum chamber. The operation process during projection is as follows. A piston (10.0 mm thick, 9.0 mm diameter) 3 made of silicon is inserted into the cylinder inlet (7.9 mm) (4) and the bottom of the cylinder is fitted with a particle carrier (6). Insert the particle projection socket (7) into the This whole part is again inserted into the air valve socket (2). When the solenoid is opened, the gas pushes the silicon piston 3 and passes through the cylinder (40.5 mm in length) to propel the particle carrier 6 attached below. It reaches the stop net at the bottom of the particle projection socket and projects only particles, and gas is diffused through the gas outlet 8 into the chamber.

3 shows the internal structure of the particle projection socket in detail. The particle projection socket is connected to the cylinder by the spiral 1 of the connecting portion. The end of the cylinder 3 is 22.0 mm in diameter. The center is 0.5mm deep 20.0mm in diameter and has a 1mm jaw around the periphery, so it is well positioned when the particle carrier 4 is attached to it. The particle carrier uses a cellophane with a thickness of 0.04 to 0.05 mm and a diameter of 19.9 mm, and when it is attached to the cylinder outlet, first, a little thin surfactant is applied to the cylinder cross section, and the side where the particles are attached is visible.

The inner diameter of the particle projection socket is equal to the outer diameter of the cylinder. At the bottom center is a particle projection port 5 having a diameter of 8.0 mm. The inside of the particle projection sphere has a circle having a diameter of 16.0 mm and a depth of 1.0 mm, where a suspension net 2 made of stainless steel having a thickness of 0.6 mm and a diameter of 15.7 mm is placed. This network serves to stop the particle carrier. The distance from the end of the cylinder to the stop net is 9.0 mm. The thickness from the end of the net to the base is 3.0 mm. The outside diameter of the particle projection socket is 32.1 mm and the height is 17.9 mm. There are 13 holes, 4.4 mm in diameter, which are just above the inner underside.

4 is an accessory for the particles to propagate with the gas to reach the target. When using, insert the particle spray kit of Figure 4 (a) into the particle projection socket as shown in Figure 4 (b). In this case, the piston, the particle carrier and the stop net used in FIG. 3 are not used. In order to use, remove the cylinder from the particle projection socket with a pipette of DNA + particle suspension on the stainless steel mesh (1), reassemble the cylinder, and insert the whole into the air valve socket. This case is similar to the PIG mentioned above and has the same advantages and disadvantages. Fungi or bacteria cultured in the medium are in close contact with the medium and are not splashed by the projection pressure, which is the most economical and convenient method for these targets. It is designed to be used for the same type, so the range of use becomes wider.

Compared with the existing device already developed for the same purpose of the effect of the present invention is as follows.

PDS-1000는 가장 상품화에 성공한 장치이지만 본 발명은 다음 두가지에서 이것보다 진보되었다. 첫째, 압력발생기작에서 Bio-Rad의 것은 가스의 압력이 어느 수준에 도달되면 파열막(rupture disk)이 파열되고, 이때 분출된 압이 입자운반체를 추진시킨다. 그러므로 투사압력의 조절은 두께가 다른 파열막을 사용해서만 가능하다. 이에 대하여 본 발명은 파열막이 없고, 압력의 조절은 조절기(regulator)로 쉽게 한다. 둘째, Bio-Rad의 것은 파열막과 입자운반체(macrocarrier)를 장착할 때 우선 이들을 케이스에 넣고 이것을 다시 진공실의 본체에 부착시키므로 과정이 매우 복잡하고 손놀림이 많아야 한다. 그러나 본 발명은 모두가 1단계의 동작으로 되므로 장치의 구조와 작업과정이 단순하다.1) Biolistic of Bio-Rad

The PDS-1000 is the most successful device, but the present invention is more advanced than this in two ways. First, in the pressure generating mechanism, when the pressure of the gas reaches a certain level, the rupture disk is ruptured, and the ejected pressure propels the particle carrier. Therefore, the adjustment of the projection pressure is possible only by using a burst film of different thickness. In contrast, the present invention has no rupture membrane, and the pressure is easily controlled by a regulator. Second, Bio-Rad's process is very complicated and requires a lot of hand movements when the rupture membrane and the particle carrier (macrocarrier) are first put in the case and attached to the body of the vacuum chamber. However, the present invention is simple in the structure and the working process of the device because all of the operation in one step.

2) Secondly, Particle Inflow Gun (PIG) has a simple structure and work process and operates at low pressure, which reduces the cost. However, the major disadvantage is that when the particle is projected onto the target cell, the gas pressure is applied to the target cell. It's going crazy, so the target flies away. To prevent this, a wire mesh may be covered on top of it, but this method is limited in its use.

3) Gene Gun II (Kwon, Seok Yoon et al., 1996) is used in Korea only in Korea, but the number is not large. This is because the particle projecting body proceeds under pressure in the gas pressure transfer method, so that the particle projecting body can be easily attached and detached (Chang Sang, 1996). The first is the problem of flying samples, as in the PIG. A bigger problem is that the wires are seeded on the sample and the particles do not penetrate the cells even at the maximum pressure of 35 kg / cm 2, which the machine allows. Secondly, the projected particles were carried on the head of the propellant made of silicon, and the particles were transported through the cylinder and buried inside the propellant and the cylinder. This causes not only the loss of the prepared particles, but also the problem that the particles wear the cylinder walls. Thirdly, the projected particles do not spread around the center of the projection, but laterally, and the falling positions are different each time, so that the particles cannot be received at the desired point of the sample. This is because the progress of the particle carrier is unstable.

The present invention is considered to be a novel invention because all of the problems pointed out in the above several different devices have been solved. That is, the structure of the device is simple compared to the existing device. ② The work process is simple. ③ The projection position is constant and wide. ④ Simple pressure control. Apart from this, particle projection kits produce high effects with low gas pressure because the particles are projected directly onto the target.

This invention is used as a tool for injecting DNA in the work (transformation) of injecting DNA (genes) of a pseudonym into plant cells or tissues and changing the traits of the cells. This was initially done by the power of bacteria to invade plants in nature, but a mechanically viable device was developed by Klein et al. (1987). This is commonly referred to as particle bombardment because it coats DNA with fine tungsten or gold particles and pushes them into the target cell with high pressure.

PDS-1000/He라는 이름으로 개발되었고 (Kikkert, 1993), 이것은 곧 Bio-Rad사로 인계되어 Biolistic^TMPDS-1000/He 라는 이름으로 생산되었다. 90년대 이후 유전자를 삽입하는 연구가 많이 이루어지면서 이 기구는 매우 유용한 것으로 인식이 되었다. 그러나 기구 자체에 비하여 값이 매우 비싸서 사용에 제한이 있을 뿐만 아니라 조작하는데 시간이 많이 걸리는 단점이 있다.Various attempts have been made to use explosives, strong voltage discharges, high-pressure nitrogen gas, compressed air and high-pressure helium gas as a means to propel particles, but helium has proved to be the most effective. High pressure (60 ~ 70㎏ / ㎠) helium gas propulsion device is biolistic by DuPont of USA

It was developed under the name PDS-1000 / He (Kikkert, 1993), which was soon taken over by Bio-Rad and produced under the name Biolistic ^TM PDS-1000 / He. Since a lot of research has been conducted on gene insertion since the 1990s, the instrument has become very useful. However, the price is very expensive compared to the mechanism itself, there is a limitation in use as well as a long time to operate.

Later, Takeuchi et al. (1992) found that the particles penetrated the target cells even when the particles were projected with helium gas of 6-7 kg / cm 2, which was lower than the electrons. Finer et al. (1992) and Vain et al. (1993) ) Developed an improved Particle Inflow Gun (PIG). This device is the use of gas pressure Biolistic ^TM PDS-1000 / He, and the economy is not only one-tenth, the structure is simple and operation is also simple to process than the high point Biolistics ^TM PDS-1000. However, there is one big drawback. It disperses the target because the particles are in intensive contact with the target with the gas. To prevent this, the wire mesh is seeded and projected onto the target, but cells smaller than the mesh of the wire mesh fall through and fly away. This is convenient when the target is stuck to the medium and is not flying under pressure.

Air is used instead of helium as the fighter's power. Oard et al. (1990,1993) made a device for accelerating tungsten particles with compressed air, and Morikawa et al. (1994) developed an apparatus for propelling a projectile by ejecting compressed air at a high pressure of 200 kg / cm 2 or more. Various types of particle accelerators have been devised, but Biolistic ^TM PDS-1000 / He is the best in terms of function. So far, only this has been produced as a commodity.

The present invention improves the disadvantages of the existing Bio-Rad products and the disadvantages of PIG, so it is considered to be the most economical, effective and popular particle projector.

Claims (5)

As a tool used to insert DNA (gene) into plant cells, when a piston (silicon ring) in a cylinder is pushed by a high-pressure gas or air force, the piston then propels the particle carrier to the stationary network, where the particles A device that is intended to be projected on. Inventive method using the structure and the desorption principle of the air valve in the method of inserting and removing the cylinder in the socket to receive the gas flowing in claim 1. The particle carrier is attached to the end surface of a cylinder of Claim 1, and the material of this particle carrier is OPP film. The pressure blocking plate is inserted under the particle projection socket, and the space above and below the plate is connected by a hose (or pipe) outside the vacuum chamber. A device in which the particle suspension is directly projected to a target under gas pressure by using a particle spray kit instead of using the piston, the particle carrier and the stop network of claim 1.

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