AU635372B2 - A method for gene transfer and a particle gun for such a method - Google Patents

Description

635372 S F Ref: 150976 FORM COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 COMPLETE SPECIFICATION

(ORIGINAL)

FOR OFFICE USE: Class Int Class Complete Specification Lodged: Accepted: Published: Priority: Related Art: *000 0 0 @0 0 00 *0 0 0000 Name and Address of Applicant: Institute of Biophysics Academia Sinica Zhong Guan Cun Hai Dian Qu Beijing 100080 PEOPLES REPUBLIC OF CHINA Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia Address for Service: n 00 0** 0 0 0 oooooo Complete Specification for the invention entitled: A Method for Gene Transfer and a Particle Gun for such a Method The following statement is a full description of this invention, including the best method of performing it known to me/us 5845/3 Abstract A method for gene transfer and a particle gun for such a method wherein a macroprojectile is placed in front of a gunpowder bullet in said particle gun, metal microparticles coated with genes from an external source are carried on the front end of said macroprojectile, upon ignition of said gunpowder bullet, said macroprojectile is impelled along a passage through a barrel, a pressure release tube, a pressure release connector and impacts on an impact plate, said microparticles inject into cells and tissues "f animals or plants, the bombardment velocity is adjustable, said bombardment can be done not only for cells and tissues in vitro in *a vacuum ultraclean table of 0.1 atmosphere but also for cells and

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*'tissues in vivo in natural environment; and said particle gun is a movable and portable particle gun, said impact plate (13) has a cone-shaped aperture, a screen element (15) is fixed at the end with larger diameter of said aperture, a sample chamber (18) is *connected hermetically to said end by a screw-thread, or the 000 components of the bombardment system are fixed inside of a muffler 'tube a sample connector (31) with a sieve (33) is connected to its lower end.

-1- FPME901426A A Method for Gene Transfer and a Particle Gun for Such a Method Background of the invention The present invention is directed to a method for gene delivery in biological genetic engineering and to the apparatus for this method, in particular to the method and specialized equipment to realize the introduction of genes from an biological source into an acceptor.

Rapid advances are now taking place throughout the world in the research to alter crop characteristics by means of genetic Sengineering. This objective is being realized by scientists through the introduction of genes into the plant organism.

According to a literatur disclosed by the British magazine "Nature SVol.327, P70, 1987" researchers of Cornell University in the U.S.A.

have described a new kind of gene transfer method and the related .*particle gun apparatus (also called gene gun, microprojectile gun). This "high velocity microprojectile method", similar to .*"nicroinjection, employs the principle of direct mechanical force ':***~enetration of plant cell walls and membranes to deliver genes from an external source into the cell by a high velocity microproje ;r~l process genes from the external source are containA 1-t the surface of metallic microparticles and injected into che _21 -I by a bombardment process). This "high velocity microprojectile process" can be regarded theoretically as a most effective gene transfer method which has very wide applications. The literatur mentioned above also disclosed a type of particle gun already developed that has successfully introduced genes from an external source into a biological tissue cell acceptor without the restrictions imposed by host limitations. From this literatur it can be learned that this type of particle gun is only suitable for laboratory oreration, requiring particle bombardment of the in vitro plant tissue be carried out in complete vacuum, and that moreover the particle gun has only a fixed velocity. In addition, :'no description of the particle gun's structure and method of its '*operation are given. From the main specifications disclosed for this particle gun the single velocity, unidirectional particle .vacuum-sealed bombardment velocity is 430m/s and is non-adjustable. As regards the particle gun, its main purpose is to perform the delivery of genes from an external biological source into an acceptor, so that the maximum number of particles are Sinjected into the cell whilst at the same time the minimum amount of damage is caused to the cell by the shock waves etc. Since the tell walls of different kinds of acceptor tissue cells have different thicknesses and strengths, and the cells themselves are differei-r sizes and shapes, the necessary particle bombardment *..v.elocities should also be different, but this is difficult to achieve with a particle gun of non-adjustable velocity, Moreover, such a type of single velocity unidirectional vacuum-sealed particle gun has stringent design demands and its operation is -2restricted to an ultraclean table. Since the particle velocity is fixed, particle impact is difficult to control, and there is no mechanism in the path from the particle source to the biological sample to prevent shock waves, fragments and other impurities from directly damaging the sample acceptor). Moreover, this type of particle gun is used for long periods on the ultraclean table, causing pollution of the table by gunpowder and other kinds of dust and destroying the aseptic working environment of the ultraclean table, as well as impairing the health of the operator. To overcome the above drawbacks of the operation of this particle gun on an ultraclean table, higher demands must be made on fabrication S":'and design, thus the overall cost will be increased. Up to now, *the particles fired from the particle gun are nonuniformly *'tistributed over the surface of the target plane, and deviation off *:*.'target occurs. This is due to the fact that the particle gun ovo-design lacks some form of target aiming and particle distribution device. The impact plate of this particle gun is inserted and withdrawn for replacement along a radial direction which is liable eb to cause the impact plate aperture to be no longer concentric with the particle gun firing line, thus when the particles traverse the .:0.impact plate it is very difficult to ensure accurate aim at the target. Another obvious drawback to this type of particle gun is its method of particle delivery is only suitable for plant issue cells in vitro and cannot be used for bombarding entire animal or plant specimens in vivo.

Summary of the invention -3- The purpose of the present invention is to overcome the defects anddrawbacks of the above mentioned gene transfer method and existing particle guns. The present invention provides a new physical process to transfer genes and the particle gun for this type of process. The particle gun of the present invention overcomes the problems of non-adjustability of the particle velocity and too stringent demands on the working environment, so that bombardment of entire in vivo samples of animals and plants in the natural state may be performed as well as bombardment of in vitro biological cells in sealed vacuum state.

According to a first embodiment of this invention, there is provided a method for qene transfer realized by particle gun, and characterized in that: a macroprojectile is placed in front of a gunpowder bullet in said particle gun, metal microparticles coated with genes from an external source are carried on the front end of said macroprojectile, upon ignition of said gunpowder bullet, said macroprojectile is impelled along a passage through a barrel, a pressure release tube, a pressure release connector and impacts on an impact plate, said metal microparticles inject into cells and tissues of animals or plants, passing through a cone-shaped aperture of said impact plate and through screen element, the velocity of said macroprojectile depends on the weight of gunpowder filled in it, proportions of said gunpowder and filler powder and its position in said barrel, said bombardment can be done not only for cells and tissues in vitro in a vacuum ultraclean table of 0.1 atmosphere but 25 also for cells and tissues in vivo in natural environment, fixing a vacuum sleeve on the outside of said barrel and connecting a movable sample chamber.

According to a second embodiment of this invention, there is provided a particle gun for use in said method for gene transfer of the first embodiment, comprising a gun body, a trigger, a safety catch, a firing pin and a barrel, and characterized in that: said particle gun is a movable and portable particle gun, its muzzle is connected to said pressure release tube by means of a 5 screw-thread, another end of said pressure release tube is connected to a connecting sleeve by means of an inner screw-thread, fixing next to said pressure release connector, said pressure release connector is fixed next amg/1452v to said impact plate successively, said impact plate has a cone-shaped aperture, a screen element is fixed at the end with larger diameter of said aperture, a sample chamber is connected hermetically to said end by a screw-thread.

The technical measures adopted to achieve the purpose of the present invention con''ert the original existing kind of particle gun from a fixed position type to a portable type, hence extending the field of application, Apart from certain changes to the gun barrel to suit necessary requiremer's, the other parts of the particle gun of the present invention such as the gun body, trigger, safety catch and firing pin all basically resemble the components of an ordinary handgun or the nail shooting gun used in construction building.

This type of portable particle gun can be inserted into an accessory vacuum chamber as part of fixed model particle gun apparatus, or the gun body can be adapted to use other forms of control for its firing system and incorporated into a fixed model particle gun apparatus.

5 o* amg/1452v Another technical measure adopted to achieve the purpose of the present invention by improving the effectiveness of gene transfer is the introduction of the multispeed concept in the particle gun of this invention, to fulfil the necessity of using different particle bombardment velocities for acceptor tissue cells of different wall thickness and strengths as well as different sizes and shapes. In the particle gun of the present invention there is a multiple choice of particle velocity for the acceptor cell tissue to be bombarded, the velocity range being 350-680m/s.

The technical measures adopted to achieve the purpose of the *;-:*present invention also include the central alignment of the barrel ,*."parts, the pressure release tube, the pressure release connector *'and the impact plate aperture in the particle gun of the present f invention, the concentricity of these parts being ensured by .X:mechanical precision. Consequently, in the actual process of particle bombardment of the target object, the center of bombardment basically coincides with the center of the target, and use is made of the high-speed energy of the particles. As a esult, the accuracy of each successive bombardment is very high, '~ensuring a deviation of less than 2mm. The central alignment accuracy of this particle gun is thus a special feature of this invention.

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The particle gun of the present invention consists of the barrel parts, macroprojectile, pressure release tube, pressure release connector, impact plate, screen elements, vacuum seals and washers, sample chamber, vacuum chamber and other elements. The gun barrel parts include front and rear barrels, between which the macroprojectile is located in a predetermined position. The gun barrel is connected to the pressure release tube, and then to the pressure release connector, impact plate and screen elements in that order. A vacuum seal and sealing washer are used to connect the particle gun to the sample chamber, as well as to locate this whole connected section in the vacuum chamber. When the trigger is pulled the firing pin hits the powder cartridge in the front of the barrel, the gunpowder blasts the macroprojectile giving it energy and a certain velocity. The macroprojectile passes down the ,pressure release tube, past the pressure release connector and hits .the impact plate. The violent collision between the highly e*ccelerated macroprojectile and the impact plate causes the metal :.:..:icroparticles on the front of the macroprojectile to pass through the small aperture in the impact plate, whilst the macroprojectile' itself disintegrates on impact and exits through the small holes of pressure release connector or the longitudinal slots of the pressure release tube. After traversing the aperture and .*"one-shaped opening of the impact plate the particles diffuse, pass through the mesh screen elements which reduces the shock wave and filters out macroprojectile fragments and scatters the metal mnicroparticles so that they are uniformly delivered into the animal or plant cells in the sample chamber.

The macroprojectile designed in the present invention is in the form of a bottle stopper and may be made of plastic, nylon or some other substance. The front end of the macroprojectile carries metal microparticles which may be made of tungsten or some other heavy metal and which carry biological genes from an external source. After the macroprojectile has been fired these metal microparticles penetrate into the animal or plant cells with a certain velocity, thus accomplishing the delivery of genes from an external source and attaining the objective of transforming the animal or plant characteristics. The average diameter of the metal microparticles ranges from 0.6 to 4im.

The technical measures adopted to achieve the purpose of the present invention include adjustment of the velocity of the .".bombarding particles by two means. One way to change the particle .velocity is to change the composition ratio of the powder mixture in the powder cartridge. For example, to obtain a given velocity it is only necessary to change the composition ratios of the gunpowder and filler powder. The gunpowder in the powder cartridge the commonly used ordinary gunpowder; the filler powder can be arbon and general purpose gunpowder filler. For example, for a S."rmacroprojectile of diameter c 6.35mm, 300mg gunpowder or 70 90 gunpowder with 30 10% gunpowder filler gives a velocity of 56Cm/s; 200mg gunpowder or 60 80% of gunpowder with 40 filler gives a velocity of 450m/s; 400mg gunpowder or 100% o gunpowder gives a velocity of 680m/s. Another way to change the particle bombardment velocity is to change the ratio of the lengths of the front and rear barrels so that the macroprojectile carrying the microparticles is located at different distances from the -7powder cartridge. By suitable selection of the gunpowder composition ratio and the relative lengths of the front and rear barrels so that the macrop:rojectile carrying the microparticles is located at different distances from the powder cartridge. By suitable selection of the gunpowder composition ratio and the relative lengths of the front and rear barrels, not only can any velocity between 350 and 680m/s be contained, but also the fabrication cost of the particle gun is lowered, the gun mechanism simplified and commerical production of the particle gun facilitated.

*6 9 For different kinds of animal and plant tissue cells, different bombardment velocities give rise to different particle distribution .*."densities on the acceptor surface. Consequently, selection of the bombardment velocity has a certain influence on the effectiveness of the gene transfer. In general, by combining the choice of three gunpowder composition ratio with the choice of three front and rear arrel length ratios it is possible to obtain nine kinds of telocities in the velocity range, there being 350, 390, 430, 470, ,*510, 550, 590, 630 and 680 m/s. This is basically sufficient for the majority of animal and plant tissue cells in vitro.

The technical measures adopted to achieve the purpose of this present invention include the capability of the portable type particle gun of the present invention to bombard biological cells in vivo in a vacuum of 0.1 atm. without the need of an ultraclean table. On the other hand, it is only necessary to install a vacuum housing outside the gun barrel and connect it to the sample chamber to be able to perform bombardment on animal and plant tissue cells in vivo under natural conditions. This type of portable vacuum sealed chamber can likewise protect the sample from bacterial contamination-an advantage not exhibited by previous existing particle guns.

At the end of the particle gun of the present invention there is a mesh screen structure, that is, between the particle gun and the sample chamber there are one or more layers of metal gauze, the purpose of which is to reduce the impact damage to tissue cells caused by a portion of the compressed air shock wave and to produce 0 a more uniform distribution and better diffusion of the particles "At the target surface.

Brief description of the drawings Now the further description of the gene transfer process according to the present invention and of the particle gun based on S..it are given with the following illustrations.

Figure 1 is a schematic view of the portable particle gun according to the present invention.

Figure 2 is a side cutaway view for the barrel of the portable particle gun shown in Figure 1.

-9- Figure 3 is a part of schematic view for connecting the sample chamber with the bombardment system of the portable particle gun shown in Figure 1, according to the present invention.

Figure 4 is a side cutaway view of the portable particle gun, according to the present invention, used as a stationary particle gun.

Figure 5 is a part of a side cutaway view for a variety of the portable particle gun according to the present invention.

The schematic view of the gun body parts and the particle bombardment system of the portable particle gun in shown in Figure i. The gun body part consists of a gun body 1, a trigger 2, a afety catch 3, a firing pin 4, a gunpowder bullet 5 fixed on the base of the bore, a barrel part 8 and a macroprojectile 6. The macroprojectile 6 is placed in front of the gunpowder bullet 5 and isually is made of plastics or nylon in a shape of a bottle topper, a bombardment system includes a pressure release tube 11, pressure relaease connector 16, an impact plate 13 and a screen element 15. Other elements used to connect -he vacuum chamber are vacuum seal 9 and sealing washer 10. The side cutaway view of barrel 8 in Figure 1 is shown in Figure 2. This part includes an outer sleeve 83, a front barrel 82 and a rear barrel 81. The macproprojectile 6 is placed in front of the gunpowder bullet 5 and between front barrel 82 and rear barrel 81. The metal microparticles or the tungsten particles 7 carry the genes from an external source on their front end. The macroprojectile 6 and the internal diameter of the barrel are fitted into each other very well so that the macroprojectile 6 will seal up the barrel. The macroprojectile 6 also plays a role of carrier. The mean diameter of the metal micproparticles is usually in the range of 0.6-4 pm, one of other sizes as 0.9, 1.2, 2.0, etc. can be also selected.

The section shown in Figure 2 can also be called microparticle delivery section, namely, while pressing the trigger 2 the gunpowder bullet 5 will impel the macroprojectile 6 into the bombardment system and upon impact of the macroprojectile against the impact plate the microparticles carrying the genes from an .external source will enter the biological material.

*e Figure 3 is a part of schematic view for connecting the S."bombardment system with the sample chamber. The bombardment system includes a pressure release tube 11, a pressure release connector 16, an impact plate 13, a screen element 15 and a connecting sleeve 24. Among them the pressure release tube 11 with more than one ongitudinal slots 22 is connected to outer sleeve 83 by means of a screw-thread and the another end is connected of the connecting sleeve 24 by means of an inner screw-thread, fixing next to the pressure release connector 16. The pressure release connector 16 a several radial outlets 12 connected to corresponding outlets on connecting sleeve 24. The pressure release connector 16 is fixed next to the impact plate 13 successively. The impact plate 13 has a cone-shaped aperture 14. The screen element 15 is fixed at the end with larger diameter of the aperture. More than one layer of metal -11grid 23 with different mesh number are fixed on the screen element The accelerated macroprojectile 6 carrying the microparticles, passing through the pressure release tube 11 and pressure release connector 16, impacts against the impact plate 13. Most of the shock waves and fragments produced by the impact will be drained away through the longitudinal slots of the pressure release tube and the radial outlets of the pressure release connector. Then, the metal microparticles 7 carrying the genes from an external source and a few of the macroprojectile fragments shoot at the screen element 15 through the aperture of the impact plate 13. The Smicroparticles are filtered and scattered by the metal grid 23 fixed on the screen element, and enter the sample chamber 18 connected with the connecting sleeve. In this way the *-microparticles are impelled into the cells and tissues and the .process of delivering genes from an external source into the cells and tissues is completed.

Figure 4 is a side cutaway view for a variety of the portable .:'article gun, according to the present invention, used as a S..stationary particle gun. Here, a sealing sleeve, instead of a gun body, is used. A control panel shown in Figure 4 replaces the trigger. This kind of stationary particle gun is fixed on a vacuum chamber 20 by means of a vacuum seal 9 and a sealing washer They form a complete bombardment system of the particle gun used for delivering gene.

-12- Figiure 5 is a part of a side cutaway view for another variety of the portable particle gun according to the present invention.

It consists of the bombardment system shown in Figure 1, a muffler tube 19, a stopper 25, a sealing cap 26, an upper housing 27, rubber 0-ring 28 and 29, a lower sealing housing 30, sample connector 31, a spring clip 32 and a sieve 33. If an ultraclean table is not convenient to use, this construction can be used for delivering gene into cells and tissues in vivo. The muffler tube 19 in this construction plays a role of the vacuum chamber 20. The size of the apparatus will be reduced and the sample chamber 18 will not be used, therefore, the structure will be simplified.

'hile the bombardment system is fixed in the muffler tube, the rubber 0-ring 29 is put onto the lower sealing housing 30 which S•.fits into the impact plate 13 and pressure release connector 16 .1 0 .tightly. The pressure release connector 16 and the muffler tube 19 fit into each other tightly as well. The stopper 25 is screwed on the front end of the pressure release tube 11. The rubber 6-ring 28 is fixed on the upper housing 27. Screw the upper housing 27 :::gnto the muffler tube 19 tightly, and seal it by means of the sealing cap 26. After fixing the gun body 1 shown in Figure 1 to the front end of the bombardment system and fixing the sample connector 31 carrying the sieve 33 and spring clip 32 to the lower ""end of the lower sealing housing 30, a complete portable particle gun is constructed.

After aiming at the cells and tissues 34 in vivo and pumping the chamber down to vacuum, it is ready to bombard the sample and -13to deliver the genes from an external source into cells and tissues in vivo.

Description of the preferred embodiment A preferred embodiment and a further description of the present invention are introduced herewith.

ExtARMPLE 1 In one preferred embodiment of the present invention, a body of ordinary nail shooting gun is used as the gun body of this portable particle gun, wherein as the velocity of the bombarding particle is

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.680 m/s and the weight of the gunpowder is 400mg, the diameter of 5 gunpowder bullet is 06.35mm, the macroprojectile with the size of *7.8 x 20mm is made of nylon. The vacuum seal 9 is covered by a *5 .sealing washer 10, one end of the vacuum seal is connected to a pressure release tube 11 of 020 x 70mm. Another end of the pressure release tube 11 with three longitudinal slots is connected :to a pressure release connector 16 of 4)20 x 10mm by means of a :screw-thread. The connector 16 has eight radial outlets 12 of 4mm diameter. Its another end is connected to an impact plate 13 which has a cone-shaped aperture of 600 taper, )lmm diameter and 0.8mm depth on it. Three layers of stainless steel grid 23 are fixed on

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too a screeen element 15 which is next to the impact plate 13. A sample chamber 18 has a size of ¢60 x 55mm. After loading the sample, one end of the chamber is connected to the connecting sleeve 24 airtightly. The step is done in an ultraclean table.

Then, set the particle gun into the vacuum chamber 20 by means of -14the vacuum seal 9 and the sealing washer 10. The size of the vacuum chamber 20 is O150 x 240mm. After setting and pumping the vacuum chamber down to 0.1 atmosphere, it is ready to start the particle bombardment. Pressing the trigger, the nylon macroprojectile carrying the tungsten microparticles impacts against the impact plate and the tungsten microparticles will pass through a cone-shaped aperture and enter the cells and tissues, the genes from an external source will be impelled into the biological material. As an example, the good results achieved by using the particle gun according to the present invention are demonstrated as follows EXAMPLE 2 In a tissue transformation test of tobacco, onion, American 355CAT ginseng, wheat, rice using the CAT, GUS and NPT I gene (P e ,P PUR and PCN plasmid DNA), a transient expression with higher transformation efficiency can be obtained. The resistant callus can be sieved out from the transformation cells of rice. After the regeneration plants can be differentiated from the

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*:':resistant callus.

In another preferred embodiment of present invention, cells of the animal or plant are bombarded directly by an improved variety C, Gt J of the portable particle gun which has a structure shown in Figure While removing the sample chamber 18 and vacuum chamber 20 of the particle gun used in the above-mentioned embodiment, a muffler tube 19 is laid on sealing washer 10 and fixed. Said tube 19 is made of alloy aluminium and has a size of (52 x 110nnm. The diameter of the gunpowder bullet is 06.35mm. The weight of the gunpowder is 300mg and the velocity of the microparticles is 560m/s. A sample connector 31 carrying the sieve is set in front of the screen element. The front end of the sample connector is aimed at the tissues of animal or plant. After pumping the muffler tube 19 down to vacuum, it is ready to bombard cells and tissues in vivo to transfer the gene. The efficacy of this particle gun is essentially the same as in the above-mentioned embodiment.

In short, the process of gene transfer provided in the present invention is simple and convenient. The velocity of the particles can be controlled satisfactorily. It depends on the amount of the .gunpowder in the gunpowder bullet and on the various combination of *0 *..front nd rear barrel. According to the thickness and hardness of 00 .*the cell wall, the size and the shape of the cell, the operator 'could choose the bombardment velocity wanted in advance. So the "'cells the tissues will be penetrated by the genes from an external source without lethal damage of the cells and tissues.

Owing to the reasonable design, convenient and wide usage, the 0.00 •portable particle gun according to the present invention gets rid the deficiencies of the apparatus available at present. It could operate not only in sealed vacuum system, but also in ***%.atmosphere circumstance for delivering genes from an external :source into cells and tissues in vivo. In addition, any variety 0 based on the present invention (including the barrel parts, impact plate, screen element, pressure release tube, pressure release -16connector, muffler tube etc.) is included in the scope of description and claims of the present invention.

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Claims (16)

1. A method for gene transfer realized by particle gun, and characterized in that a macroprojectile is placed in front of a gunpowder bullet in said particle gun, metal microparticles coated with genes from an external source are carried on the front end of said macroprojectile, upon ignitiion of said gunpowder bullet, said macroprojectile is impelled along a passage through a barrel, a pressure release tube, a pressure release connector and impacts on an impact plate, said metal microparticles inject into cells and tissues of animals or plants, passing through a cone-shaped .**:*aperture of said impact plate and through screen element, the .velocity of said macroprojectile depends on the weight of gunpowder *5 "filled in it, proportions of said gunpowder and filler powder and position in said barrel, said bombardment can be done not only .:.for cells and tissues in vitro in a vacuum ultraclean table of 0.1 atmosphere but also for cells and tissues in vivo in natural environment, fixing a vacuum sleeve on the outside of said barrel *off@: S'"nd connecting a movable sample chamber.

The method for gene transfer of Claim 1 wherein said metal microparticle is tungsten or other heavy metal particle with mean diameter of 0.6-4 um. -18-

3. The method for gene transfer of Claim 1 wherein said gunpowder filled in said gunpowder bullet is ordinary gunpowder and said filler powder includes carbon powder and general filler powder.

4. The method for gene transfer of Claim 1 wherein said microparticle is made of plastics or nylon or other materials and is of the shape of a bottle stopper.

The method for gene transfer of Claim 1 or Claim 2 wherein said available bombardment velocity is in a range of 350-680m/s, said macroprojectile being of a diameter of p6.35mm and, filling with gunpowder of 300mg or 70%-90% gunpowder plus 30%-10% filler powder, velocity of 560m/s can be reached, filling with said gunpowder of *200mg or 60%-80% gunpowder plus 40%-20% filler powder, a velocity t.f 450m/s can be reached, filling with said gunpowder of 400mg or gunpowder, a velocity of 680m/s can be reached, combining above-mentioned weights of said gunpowder of the proportions of said gunpowder and said filler powder with three different positions of said macroprojectile in said barrel, nine different SVelocities can be reac d, namely 350m/s, 390m/s, 430m/s, 470m/s, S 10m/s 550m/s, 590m/s, 630m/s ,nd 680m/s.

6. A particle gun for po-i3A use in said method for gene transfer *pf Claim 1, comprising a gun body, a trigger, a safety catch, a iring pin and a barrel, and characteried in hat and characterized in that -19- said particle gun is a movable and portable particle gun, its muzzle is connected to said pressure release tube by means of a screw-thread, another end of said pressure release tube is connected to a connecting sleeve by means of an inner screw-thread, fixing next to said pressure release connector, said pressure release connector is fixed next to said impact plate successively, said impact plate has a cone-shaped aperture, a screen element is fixed at the end with large diameter of said aperture, a sample chamber is connected hermetically to said end by a screw-thread.

7. The particle gun for use in said method for gene transfer of claim 1, comprising a gun body, a trigger, a safety catch, a firing pin and a barrel, and characterized in that: Sgo said particle gun is a movable and portable particle gun, its muzzle is connected to said pressure release tube by means of a screw-thread, anothe: end of said pressure release tube is connected to a connecting sleeve by means of an inner screw-thread, fixing next to said pressure release connector, said pressure release connector is fixed next to said impact plate successively, said impact plate has a cone-shaped 20 aperture, a screen element is fixed at the end with larger diameter of said aperture, these components of the bombardment system are fixed inside of a muffler tube by means of a stopper, a sealing cap, an upper housing, rubber O-ring, and a lower sealing housing, to its lower end a sample connector with a sieve is connected by means of a screw-thread.

8. fhe particle gun for use in said method for gene transfer of claim 6 or 7, wherein the ratio of the lengths of front barrel and rear barrel of said barrel part is in three different values.

9. The particle gun as defined in any one of claims 6 to 8 wherein said pressure release tut-c has more than one longitudinal slots uniformly distributed thereon.

The particle gun as defined in any one of claims 6 to 9 wherein said pressure release connector has more than one radial outlet S uniformly distributed on thereon.

11. The particle gun as defined in any one of claims 6 to wherein more than one layer of metal rid with different mesh number is fixed on said screen element.

12. The particle gun as defined in any one of claims 7 to amg/1452v -21- wherein said sieve comprises more than one layer of metal grid with different mesh number.

13. The particle gun as defined in any one of claims 6 to 11 wherein a central passage is concentrical through said front barrel, said rear barrel, said pressure release tube, said pressure release connector, said impact plate and said screen element and its concentricity is ensured by mechanical precision.

14. The particle gun as defined in claim 6 or 7 wherein said movable and portable particle gun can be modified to use as a stationary particle gun, replacing said gun body and trigger by a sealed sleeve and a control panel respectively, this kind of stationary particle gun is fixed to said vacuum chamber by means of a vacuum seal and a sealing washer.

15. A method for gene transfer realized by particle gun which method is substantially as herein described with reference to Example 1 or 2.

16. A particle gun for use in the method of gene transfer of claim which gun is substantially as herein described with reference to any i" one of Figs. 1 to :DATED th,s EIGHTEENTH day of JANUARY 1993 Institute of Biophysics Academia Sinica Patent Attorneys for the Applicant SPRUSON FERGUSON amg/1452v