CN1052695A - A method for gene transfer and its gene transfer particle gun - Google Patents

Abstract

The present invention belongs to the field of the method for introducing into animal and plant cells in biogenetic engineering and the instrument for its operation, especially the method for realizing the introduction of biological exogenous gene into the recipient cells in vitro and in situ And the equipment dedicated to this method, the method of gene transfer provided by the present invention can accurately control the speed and method of bombardment according to the proportion of gunpowder, and can be operated under closed state and non-closed state with special equipment, which can be used for isolated The bombardment of somatic biological cells can also be used to bombard the original biological cells to introduce foreign genes, thereby changing the traits of organisms.

Description

The invention belongs to the field of a method for realizing gene introduction in biological genetic engineering and an instrument for its operation, in particular a method for realizing the introduction of a biological exogenous gene into a receptor and its special equipment.

At present, the research on changing the traits of crops by means of genetic engineering is progressing rapidly in the world. Scientists achieve this by introducing genes into plants. In order to achieve this goal, researchers from Cornell University in the United States use the concept of accelerating particles into biological cells. The constructed "particle gun" has successfully realized the introduction of exogenous genes with biological tissue cells as receptors without limitation of the host range. But so far in the market, there are no commercial products for sale at home and abroad. The particle gun they made in the laboratory has only been reported as having a speed and carrying out particle bombardment methods and equipment for plant isolated tissues in a fully enclosed vacuum. The structure and implementation method of their particle guns have not been reported. At present, the main indicators of the particle guns are: single-speed, one-way, fully enclosed, and the particle bombardment speed is 430 m/s, which cannot be adjusted. The main index of the actual particle gun should be that the maximum particle is injected into the cell while the damage to the cell caused by the shock wave should be minimized, and the particle gun without speed regulation is difficult to meet the above requirements, so how to control the bombardment The speed and direction of the bombardment are the biggest problems for researchers at present, because the cell walls of various recipient tissue cells are thick or thin, the degree of hardness is different, and the cells are different in size and shape, so the bombardment speed is required to be different. In addition, the technical requirements for the design of the fully enclosed gun are also complicated, which not only wastes expenses but also loses the advantage of carrying out particle bombardment on the original body of plants in situ in an unenclosed state.

references:

1. Scieme V01.244, 1989, P1293

2. Nature V01.327, 1987, P70

The purpose of the present invention is to overcome the shortcomings and shortcomings of the above-mentioned gene transfer method and particle gun, to provide a new physical method of gene transfer and a commercialized gene transfer particle gun with bidirectionality and adjustable speed specially used for this method . This gun can bombard isolated biological cells in a vacuum state, and can also bombard in-situ particles of the original body in a closed state, and can adjust the speed of the bombarded particles, variable speed from 350-460- 640 m/s.

The invention provides a physical method for transferring biological genes and a speed-adjustable two-way gene transfer particle gun dedicated to the method, which can inject microparticles coated with genes into plant and animal cells, thereby realizing exogenous The introduction of genes achieves the purpose of changing the traits of animals and plants. The particle gun of the present invention can detonate a φ6.35mm gunpowder bullet containing different proportions to achieve different bombardment speeds. A large projectile is loaded in front of the gunpowder, and the front surface of the large projectile is loaded with microtungsten particles. The gun has a detonation speed ranging from 350-640M/sec, which are 640m/s high speed, 460m/s medium speed and 350m/s low speed. It also has a two-way function that can carry out particle bombardment on isolated animal and plant tissues in a closed state at a low vacuum of 0.1 atmospheric pressure to achieve exogenous gene introduction, and can also hold a particle gun in the atmosphere to target animals and plants. In situ tissue particle bombardment to inject exogenous genes into cell tissue.

The gene transfer method of the present invention and the particle gun dedicated to the method are described below in conjunction with the accompanying drawings. The speed-adjustable bidirectional gene transfer particle gun of the present invention is composed of four major parts: (as shown in Fig. 1, Fig. 2, Fig. 3, As shown in Figure 4)

(1) Gun body (2) Particle bombardment system

(3) Sample chamber (4) Steel chamber and vacuum unit

In Fig. 1, it is shown that the gun body part and the particle bombardment system are integrated. The gun body part is a common pistol mechanism or a nail gun mechanism used in the construction industry, which includes a handle (1), a trigger (2), a safety button (3), a firing pin (4), and a On, gunpowder bullet (5), large-scale projectile (6), large-scale projectile is one of feature of the present invention, and it is contained in the front surface of gunpowder bullet (5), and it can be one section of plastics and can also be made bottle stopper with nylon type, it is inserted into the mouth of the gun barrel (8) for tight fit, its function is that it plays a role in sealing the gun barrel when the inner chamber of the gun is evacuated, and it can also play a carrier role, because there is a belt in front of it Micro tungsten particles (7) of exogenous genes, the diameter of micro tungsten particles is 0.6-4μm, put them into the gun chamber together, this part can also be called the emission part of the micro particles, that is, when the trigger is pressed, the gunpowder will be fired immediately Then send the large plastic projectile to the next part of the particle bombardment system to bombard the receptor to achieve gene introduction.

The second most of the bombardment system includes a sealing plug (9) made of steel, and its plug cap portion is fixed on the muzzle with clips, requiring a tight fit. There is a rubber sealing gasket (10) set on the top of the sealing plug (9), (the steel chamber in Figure 2 fits closely with the gasket (9)), the other end of the sealing plug (9) is connected with the air outlet ( 12) The gun barrel (11) is connected, the other end of the gun barrel (11) is fixed with the screw of the joint head (16) with several air outlets, and the other end of the metal joint head (16) is screwed with the blocking plate (13) Cooperate. There is a trumpet-shaped emission hole (14) in the center of the blocking plate (13) made of hardened steel, which is screwed with the filter plate (15), and the filter plate (15) is a steel sleeve with a thickness of 20 mm × 18 mm. There are several layers of stainless steel mesh, the purpose of which is to inject particles from the bell mouth of the blocking plate (13) and enter the animal and plant cells in the sample chamber (16) during particle bombardment, and the stainless steel mesh will filter some small fragments again , there are air outlet holes on the barrel (11) and the blocking plate (13), its function is to leak the shock wave of compressed air formed in the barrel by the large projectile after acceleration, and the other function is to use steel in the particle bombardment system When the chamber was sealed and vacuumed, they were air holes again, and the blocking plate (13) had holes through the horn emission hole (14) of φ 1.2mm-0.9mm×depth 0.6mm-1.0mm×cone angle 50°-70°. The blocking plate is a φ20mm × thick 10mm steel plate made of hardened steel. The effect of the blocking plate (13) is to prevent the passing of the large projectile after the detonation of the first stage, and to relieve the projectile from flying due to the compression of the gun barrel. The shock wave that air forms, gun barrel (8) is to be made by φ 20mm * long 70mm steel pipe, has some ellipse-shaped air outlets above and can leak most of shock wave. When carrying out fully enclosed state operation, promptly steel chamber (17) is enclosed within on the sealing washer (10) on the gun and fixed, the internal diameter of steel chamber determines the size of sealing washer (10), and they will closely match. Before putting on the steel chamber, first install the isolated animal and plant samples to be bombarded in the sample chamber (18). The sample is placed in the center of a metal petri dish, and the size of the petri dish is just fixed on the fixed base in the sample chamber, so as to achieve The positioning is accurate. Then fix the sample chamber (18) on the filter plate (15), and then connect the air nozzle (19) of the steel chamber (17) to the vacuum pump, and then the bombardment system and the gun body of the particle gun can be evacuated , when bombarding the original body of animals and plants in situ, it is not necessary to work in a closed state, so that the steel chamber and the sample chamber are not put on, but a sound-absorbing sleeve (20) is placed on the sealing gasket ( 10) As shown in Figure 4, the sound-absorbing sleeve is as shown in Figure 4. Its inner diameter only needs to fit closely with the sealing ring (10), and they are fixed with clips. It is generally made of metal. Its function is to Protect the safety of the operator at the same time and relieve the shock wave in the protection tube. During operation, the operator can hold the sound-absorbing protective tube (20) and aim at the original body to bombard the in-situ animal and plant samples.

Example 1:

Use the needle gun produced in Yangzhou as the main body of the transfer gun, the bombardment speed is 640 m/s, and the gunpowder ratio is 100% of the usual gunpowder to make a large projectile with a size of 6.35mm, which is nylon with a diameter of 7.8mm and a length of 20mm Bullets, a sealing plug (9) with an inner diameter of φ40mm made of steel is set on the muzzle, a rubber sealing ring (10) of φ50mm is placed on the outer surface of the sealing plug, and the other end of the sealing plug (9) has a hole in the inner diameter of φ20mm× The gun barrel (11) made of long 70mm steel pipe is tightly fitted and inserted, and three oval-shaped air outlets (12) are opened on the gun barrel (11), and the other end of the gun barrel (11) is quenched The connecting head (16) that steel is made is screw-fitted and fixed, and there are 8 air outlets of φ 4mm evenly around the connecting head (16). The blocking plate (13) has a φ1mm, a depth of 0.8mm, and a horn-shaped emission hole with a cone angle of 60°, which is made of hardened steel and connected with the coupling head (16). The bottom of the filter plate (15) is fixed with three layers of stainless steel mesh, the size of the mesh is just stuck in the inner diameter of the filter plate (15), the bottom sample chamber (18) is made of steel with a length of 60m×55mm, one end of which is in a spiral phase with the filter plate Cooperate, and the other end is sealed with a φ50 threaded plate after the sample is installed. Steel chamber (17) is that φ 150mm * 240mm * wall thickness 5mm has air extraction hole (19) to connect mechanical pump, mechanical pump model: 2HZ-I type rotary vane vacuum pump, vacuum meter is produced by the third factory of automatic instrument.

Example 2:

The particle gun that operates under semi-closed state, gun body (1), bombardment system (2) are all the same as embodiment 1, remove steel chamber (17) and sample chamber (18) in embodiment 1, seal The washer (10) is put on the sound-absorbing protection cylinder (20) made of aluminum alloy of φ52mm×length 110mm×thickness 6mm, and is fixed with an iron clip. The gunpowder bomb uses a φ6.35mm size, and the gunpowder ratio is 70% for general use. Gunpowder, 30% carbon powder as filler. Achieving a gunpowder velocity of 460 m/s.

The physical method of gene transfer provided by the present invention is simple and easy to implement. The speed of bombarding particles is controlled and realized according to the ratio of gunpowder bullets. This method is accurate in quantification and speed control. The thickness, thinness and hardness of the animal and plant cell walls determine the bombardment speed, and then the gunpowder ratio can be selected, so as to avoid the inability of exogenous genes to be injected or excessive impact on the recipient cells due to improper bombardment speed. Cause unnecessary loss of cells, so that the cells are damaged.

The special equipment gene transfer particle gun provided by the present invention is reasonable in design and small and exquisite. It does not need to seal the whole gun in the vacuum system, but only needs to fix a particle bombardment system on the gun muzzle. This part satisfies the bombardment operation in a closed state, changing In addition, this part does not need to be sealed, creating a special function of introducing exogenous genes in situ to the original organism in the atmosphere, which is not available in the existing technology, and will contribute to bioengineering. It provides an extremely powerful tool to realize the exogenous gene transfer technology in various organisms.

Claims (7)

1, a kind of method that adopts particle gun bombardment metastatic gene, it is characterized in that: the gunpowder bullet front at particle gun is loaded with large-scale projectile, the front surface of projectile is loaded with little tungsten particle, have foreign gene on little tungsten particle, emission bombardment by gunpowder accelerates to the micropartical that has foreign gene within the acceptor, its bombardment speed is concerned by the decision of gunpowder proportioning: adopt 640 meter per second bombardment speed with adorning 100% gunpowder in the φ 6.35mm gunpowder bullet, the weighting agent of 460 meter per second 70%-90% gunpowder and 30%-10%, the bombardment speed gunpowder proportioning of 350 meter per seconds is the weighting agent of 60%-80% gunpowder and 40%-20%, can carry out more than 0.1 normal atmosphere under closure state during bombardment; Also can in atmosphere, operate.

2, by the method for the described metastatic gene of claim 1, it is characterized in that: said gunpowder is commercially available general gunpowder, and the gunpowder weighting agent comprises carbon dust and general gunpowder weighting agent.

3, by the method for the described metastatic gene of claim 1, it is characterized in that: the diameter range of said little tungsten particle is from several the measuring of 0.6 μ m-4 μ m.

4, by the method for the described metastatic gene of claim 1, it is characterized in that: said large-scale projectile can be that general plastics can be that nylon is made.

5, a kind of metastatic gene particle gun that is exclusively used in the method for the described metastatic gene of claim 1, by the small of the stock (1), plate machine (2), safety knobs (3), firing pin (4), gunpowder bullet (5), gun barrel (8) is formed, it is characterized in that: fix a metal plug (9) that is with rubber ring joint (10) at the muzzle place, sealing plug (9) the other end is fixed on the gun barrel (11) that has production well (12), an end that has production well on the other end of gun barrel (11) and the metal connecting joint (16) links, there is a trumpet type transmitting aperture (14) in barrier plate (13) central authorities of making of quenched nickelclad, it and coupling head (16) screw fixed, the other end joins with stainless (steel) wire screen plate (15) spiral of band several layers, gunpowder bullet (5) is contained on gun barrel (8) the internal fixing seat, large-scale projectile (6) is placed on the front surface of gunpowder bullet (5), large-scale projectile (6) front surface is loaded with little tungsten particle of band foreign gene, then metal noise reduction protection tube (20) is enclosed within on the seal washer (10); Perhaps take off noise reduction protection tube (20), sample chamber (18) are fixed on and put steel chamber (17) on the screen plate (15) again on seal washer (10), and vacuum pump is by to the aspirating hole (21) of steel chamber (17).

6, by the particle gun of the described metastatic gene of claim 5, it is characterized in that: said metal noise reduction protection tube (20) can be aluminium, aluminium alloy.

7, by the particle gun of the described metastatic gene of claim 5, it is characterized in that: the screen plate of said band stainless (steel) wire (15) contains the stainless (steel) wire more than two layers at least.

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