KR100317973B1 - Ball-valve controlling device of gas or air in particle bombardment system for transformation of cell - Google Patents

Abstract

The present invention is a tool used when mechanically inserting DNA (gene) into a cell (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.

Existing representative machines are well known Biolistic PDS-1000 and Particle Inflow Gun (PIG). The biggest problem with these 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 other existing devices have been solved. This simplifies the structure of the device, making it easy to use and the target does not bounce because the incoming gas does not enter the vacuum chamber after completing the mission. In addition, it is possible to use two different devices depending on the use.

Description

Ball-valve controlling device of gas or air in particle bombardment system for transformation of cell

This invention is used as a tool for injecting DNA in the work (transformation) of injecting external DNA (gene) into a tissue or cell of an organism to change its original trait. 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.

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 of generating the propulsion of particles (Soughgate, 1998). A high-pressure (60-70 kg / cm2) helium gas-propelled device was developed by DuPont in the US under the name Biolistic PDS-1000 / He (Kikkert, 1993), which was soon taken over by Bio-Rad. have. 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.

Takeuchi et al. (1992) found that the particles penetrate the target cells even when the particles were projected with helium gas of 6-7 kg / cm2, which is lower than the previous one. Finer et al. (1992) and Vain et al. 1993) developed an improved Particle Inflow Gun (PIG). This device has advantages over BiolisticsTM PDS -1000 due to its economical, simple structure and simple operation, since the gas pressure is only 1/10 of BiolisticsTM PDS -1000 / He, but it has one major drawback. It disperses the target because the particles are in intensive contact with the target with the gas. In order to prevent this, the wire mesh is seeded and projected on the target, but a target smaller than the mesh of the wire mesh, such as a single cell, falls through the wire mesh and is blown away, and particles are caught in the wire mesh, so the projection efficiency is low. This is convenient when the target is stuck to the medium and will not bounce under pressure.

Air is used instead of helium as the fighter's power. Oard et al. (1990,1993) created a device that accelerates tungsten particles with compressed air, and Morikawa et al. (1994) developed a device that propels a projectile by ejecting compressed air at a high pressure of 200 kg / cm2 or more. Although the type of particle projector was devised, BiolisticsTM PDS -1000 / He is the best in terms of function, so far it is the most sold as a commodity. I have a lot of inconvenience in using these devices, I developed them and other new devices and applied for a patent in 1997 (application number: 97-007933)

Since then, all the previous models had to insert the pressure barrier plate every time they were projected, so I developed a new model that eliminated it. This exits through a separate discharge line before the gas exits the vacuum chamber. Along with this feature, several improvements have been filed for another patent on the same device.

1 is a perspective view showing the overall model of the invention

Figure 2 is a vertical view of the piping room installed on the top of the device

Fig. 3 is a longitudinal section of the gas tank 3-1, the disc holder 3-2 and the stop network holder 3-3.

Fig. 4 is a longitudinal section when the particle spray network and it are mounted on the particle propellant;

The part with the door 4 in FIG. 1 is used as the vacuum chamber 10 (inner 15x15x25cm). The vacuum is for promoting the scattering of the particles and the use range is about 20 ~ 40hg / cm2. All sides of the box wall are made of transparent PC board, and all sides except the front door (4) are covered with 2mm steel plate to withstand the pressure generated when high pressure gas is ejected. At the center of the left and right sides and the back of the vacuum box, respectively, a light emitting window 5 is placed to brighten the inside of the vacuum chamber.

The upper part of the apparatus is a piping chamber 8 (Fig. 2), which is provided with a main part related to particle projection and air ingress and discharge, and a pressure gauge 1 and a vacuum gauge 2 are attached to the front thereof. The pressure gauge indicates the proper pressure of the gas used for the projection and the vacuum gauge indicates the degree of vacuum in the vacuum chamber. Air discharge of the vacuum chamber is made through the vacuum chamber air outlet 15 and the vacuum motor connecting pipe (17). The gas which enters through the gas inlet pipe 9 from the outside of the apparatus enters the gas tank 22 via the gas inlet 11 by adjustment of the three-hole ball valve 13. When the knob of the ball valve is turned clockwise, the gas enters the gas tank 22, and when it is turned to the original position, the inflow of gas stops. When it is turned counterclockwise from the original position, the gas which has entered inside escapes through the air filter 14.

In FIG. 3, when the gas pressure of the gas tank 22 reaches a certain level, the bursting disk 24 bursts and the ejected gas propels the particle transport disk 26 past the particle propagation pipe 25. At this time, the particles 27 also proceed to reach the stop network 29. Here, the particle carrier is caught in the stationary network, and only the particles escape through the network to penetrate the cell, which is the target (7). The gas cannot proceed further in the future because of the particle carrier and is reversed through the gas outlet 18 and discharged out of the apparatus.

A device for introducing genes into cells using the particle projection method has already been developed, but the present invention is significantly different from those of the present invention. It is exiting in the reverse direction when it comes in. The effect of this is to keep the vacuum level in the vacuum chamber to the end, and the sample does not move, which is very stable. Secondly, the present invention can replace the high-pressure solenoid valve used for the gas inlet and outlet control in the existing product with a ball valve, the structure is simple and economical. Third, the functions of the two existing devices, Biolistic PDS 1000He and PIG gene gun, can be used as one device.

Claims (3)

In an apparatus for bombarding DNA (gene) coated fine particles to cells or tissues by a high pressure gas or air force, a rectangular parallelepiped composed of a pipe chamber 8 on the upper side and a vacuum chamber 10 on the lower side ( As the apparatus of Fig. 1, the piping chamber has a gas inlet 11, a gas outlet 18, and a pressure gauge connector 19 in the center thereof, and a vacuum chamber air inlet 10, a vacuum chamber air outlet 15, and a vacuum gauge in the periphery thereof. There is a connecting pipe (16), the gas induction pipe (9), the vacuum motor connecting pipe (17) on the rear side, the two-ball ball valve (12) and three-ball ball valve (13) for controlling the inflow and outflow of gas in the device A pressure gauge (2) and a vacuum gauge (1) are attached to the front surface, and the vacuum chamber at the bottom of the apparatus is separated from the piping chamber by the panel 20, and connected through the center of the panel (Fig. 3-1). There is a gas entry path 21 and a gas tank 22, and a separate disk holder (Fig. 3-2) is connected to the bottom of the gas tank, where Six gas outlets 18 connected to it of the tank are bent outwardly, and there are grooves in which the particle propulsion tube 25, the rupture disk 24, and the particle carrier disk 26 are located, respectively, below the disk holder. Particle projection device, characterized in that the stationary network holder (Fig. 3-3) is connected and there is a place for placing the stationary network The particle projection apparatus according to claim 1, wherein the particle transport disk (26) and the particle spray network (31) are used together or only the particle spray network (31) is used (FIG. 4). The particle projection apparatus according to claim 1 or 2, wherein the inflow and outflow of gas and air from the outside of the apparatus is controlled by ball valves (12, 13) located in the piping chamber (Fig. 2).