CN1052695A - Transfer genes method and particle gun - Google Patents

Transfer genes method and particle gun Download PDF

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Publication number
CN1052695A
CN1052695A CN 89109334 CN89109334A CN1052695A CN 1052695 A CN1052695 A CN 1052695A CN 89109334 CN89109334 CN 89109334 CN 89109334 A CN89109334 A CN 89109334A CN 1052695 A CN1052695 A CN 1052695A
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powder
bombardment
gene
particle gun
barrel
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CN 89109334
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Chinese (zh)
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姚山麟
刘仲岚
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中国科学院生物物理研究所
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Publication of CN1052695A publication Critical patent/CN1052695A/en

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Abstract

The invention is used in biological genetic engineering to induce external gene into the isolated or original cell. The technique and equipments are present in the invention. The bombardment velocity of gene is controlled accurately according to the direction of gunpower. The operation is conducted in closed or unclosed condition with special tools to induce the gene into isolated or original cell to change the character of plant or animal.

Description

本发明属于生物遗传工程中实现基因导入的方法及其操作用的仪器领域,特别是用来实现生物外源基因导入受体的方法及其专用设备。 The present invention resides in a method of genetic engineering and gene transfer achieved by the operation of the field instruments, in particular to implement the methods foreign gene into a biological receptor and its special equipment.

目前世界上用遗传工程手段改变作物性状的研究进展迅速,科学家们通过把基因导入植物体内来实现的,为了达到这一目的,美国康乃尔大学的研究者们利用加速粒子射入生物细胞的概念构造的“粒子枪”成功地实现了以生物组织细胞为受体,而无宿主范围限止的外源基因导入。 Progress is currently the world's changing crop traits with genetic engineering means fast, the scientists achieved by introducing genes into plants, in order to achieve this goal, the United States Cornell University researchers used to accelerate particles launched into the concept of biological cells configuration "particle gun" successfully achieved foreign gene into recipient cells to a biological tissue, without limiting, the host range. 但是至今在市场上国内、外还未有商品出售,他们在实验室做的粒子枪只报道过是具有一种速度的和在全封闭真空中对植物离体组织进行粒子轰击方法和设备,从未报道过他们粒子枪的结构及实施方法,目前粒子枪主要指标:单速单向全封闭型的,粒子轰击速度为430米/秒,不可调节。 But so far in the domestic market and foreign trade have not been sold, they do in the laboratory of particle gun is only reported to have a speed and in vitro tissue of plants in closed vacuum particle bombardment methods and equipment, from not been reported their structure and particle gun method embodiment, the current particle gun primary: unidirectional single-speed fully enclosed type, particle bombardment speed of 430 m / sec, can not be adjusted. 而实际粒子枪主要指标应是最大值的粒子射入细胞内的同时由冲击波等带来对细胞的损害要降至最小,而不的调速的粒子枪很难达到上述要求,因此如何控制轰击的速度及方向是目前研究者的最大问题,因为各类受体组织细胞的细胞壁有厚有薄,坚硬程度不同,和细胞有大有小、形状也不同所以要求轰击速度不同。 The main indicators of the actual particle gun should be the maximum particle injection at the same time within the cell by the shock wave caused damage to the cells should be reduced to a minimum, and not the speed of the particle gun is difficult to meet these requirements, and therefore how to control bombardment the speed and direction of the biggest problem is the researchers, because the cell wall of various types of tissue cells have receptors thick thin, hard different degrees, and cells have different sizes, different shapes so that a different speed requirements bombardment. 此外全封闭型枪设计上技术要求也复杂,既浪费了开支又丧失了在非封闭状态下对植物的原体原位进行粒子轰击的优越性。 Also fully enclosed gun design technology requirements are complicated, only a waste of expenses and lost the superiority of the original plants in situ particle bombardment carried out in a non-closed state.

参考文献:1、Scieme V01.244,1989,P12932、Nature V01.327,1987,P70本发明的目的在于克服上述转移基因方法及粒子枪的缺点和不足,提供一种新的转移基因的物理方法及专用于该方法的具有二向性、可调速的商品化转移基因的粒子枪。 References: 1, Scieme V01.244,1989, P12932, Nature V01.327,1987, P70 object of the present invention is to overcome the above disadvantages and drawbacks of gene transfer and particle gun method, there is provided a novel method of physical gene transfer and specific to the commercial process has a dichroic adjustable speed transfer particle gun gene. 这种枪可在真空状态下对离体生物细胞进行轰击,也可在封闭状态下对原体原位粒子进行轰击的功能,并可以调节进行轰击粒子的速度,可变速度从350-460-640米/秒。 Such guns may be performed on cells in vitro biological bombardment in a vacuum state, the function may be performed in situ bombardment of the original particles in the closed state, and can adjust the speed of bombarding particles, from the variable speed 350-460- 640 m / sec.

本发明提供的一种转移生物基因的物理方法及其专用于该方法的可调速二向转移基因粒子枪,它能把外面包有基因的微粒子射入植物、动物细胞内,从而实现外源基因的导入,达到改变动、植物性状的目的,本发明的粒子枪可引爆一个φ6.35mm大小,含不同配比的火药弹来实现轰击速度的不同。 Physical method of transferring a gene of the present invention provides a biological and may be specific to the method of particle gun to transfer speed two genes, which genes can microparticles wrapped incident plants, animal cells, thereby achieving the exogenous introduced genes, the purpose of dynamic change, plant traits, particle gun according to the present invention may be a detonating φ6.35mm size, containing different proportions of powder projectile bombardment to achieve different speeds. 在火药弹前载有大型射弹,大型射弹前表面载有微钨粒子。 Before the powder has a large missile projectile, the surface carrying the micro-particles before the large tungsten projectile. 该枪具有引爆速度从350-640M/sec范围,分别为640米/秒高速,460米/秒中速、350米/秒低速。 Detonation gun has a velocity from 350-640M / sec range, namely 640 m / s speed, speed 460 m / sec, 350 m / sec low. 它又具有双向即可在封闭状态下在低真空0.1个大气压以上即可对离体的动、植物组织进行粒子轰击实现外源基因导入,也可以手持粒子枪在大气中对着动、植物原体原位组织进行粒子轰击将外源基因射入细胞组织内。 It can be a two-way can be in the closed state of the animal and plant tissue in vitro is to achieve particle bombardment exogenous gene, it may be hand-held gun to move the particles in the atmosphere above the low vacuum 0.1 atm, the original plant situ tissue particle bombardment incident foreign gene within cells and tissues.

下面结合附图对本发明的转移基因方法和专用于该方法的粒子枪进行说明,本发明的可调速双向转移基因粒子枪由四个大部分组成:(如图一、图二、图三、图四所示) DRAWINGS method for transferring a gene specific to the present invention and the method of particle gun will be described, can speed bidirectional gene transfer particle gun according to the present invention consists of four major components :( Figure 1, Figure II, Figure III, FIG. IV)

(一)枪体 (二)粒子轰击系统(三)样品室 (四)钢室和真空机组在图一中表示枪体部分和粒子轰击系统做成一体。 (B) particle bombardment system (a) the gun body (iii) a sample chamber (D) and the vacuum chamber unit represented steel gun and particle bombardment portion integrally formed in a system of FIG. 枪体部分是通常的手枪机构或建筑行业用的射钉枪机构,它包括枪把(1)、板机(2)、安全钮(3)、击针(4)、安装在枪膛固定座上的、火药弹(5)、大型射弹(6),大型射弹是本发明特征之一,它装在火药弹(5)的前表面,它可以是一段塑料也可用尼龙做成瓶塞型,把它插在枪管(8)口内紧密配合,它的作用在于当枪内膛抽真空时它起到密封枪管的作用,另外它可以起到载体作用,因为在它前面载有带外源基因的微钨粒子(7),微钨粒子的直径为0.6-4μm,把它们一起装到枪膛内,这一部分也可叫做微粒子的发射部分,也就是当按下板机火药即刻发射然后把大型塑料射弹发送到下一部分粒子轰击系统内对准受体轰击实现导入基因。 Gun body portion is generally pistol nail gun means or mechanism used in the construction industry, comprising a gun (1), the trigger (2), the safety knob (3), firing pin (4), mounted on the barrel holder the gunpowder shells (5), a large projectile (6), is one of the major features of the projectile of the present invention, which is mounted on the front surface of the powder shells (5), which may be a piece of plastic can also be made of nylon stopper type, it is inserted in the barrel (8) close fit in the mouth, its role is that when the gun bore which acts as a vacuum seal the barrel, while it can play the role of a carrier, because it contains the band in front of micro exogenous gene tungsten particles (7), the diameter of the micro particles of tungsten is 0.6-4μm, put them together within the chamber, this part of the emission portion can also be called the fine particles, i.e. when the trigger is pressed immediately transmit gunpowder the projectile is then transmitted to the large plastic receptor and the alignment part of the bombardment particle bombardment system implemented introduced gene.

第二大部分轰击系统包括用钢材做成的密封塞(9),它的塞帽部用卡子固定在枪口上,要求紧密配合。 Most bombardment second system comprises a sealing plug made of steel (9), that portion of the plug cap fixing clip on the muzzle, require tight fit. 在密封塞(9)的上面套有一橡皮密封垫圈(10),(图二中的钢室与垫圈(9)紧密套上配合的),密封塞(9)的另一端与带有出气孔(12)的枪管(11)相接,枪管(11)的另一端与带若干个出气孔的联接头(16)螺旋相固定,金属联接头(16)另一端与阻挡板(13)螺旋配合。 In the sealing plug (9) above has a rubber sleeve gasket (10), (two in FIG steel chamber gasket (9) in close fitting sleeve), the other end of the sealing plug (9) and having a hole ( 12) of the barrel (11) in contact, a plurality of exit holes coupling head (16) fixed to the other end of the helical barrel (11) with the belt, the metal coupling head (16) and the other end of the blocking plate (13) spiral cooperation. 用淬火钢做成的阻挡板(13)中心开有喇叭型发射孔(14),它并与过滤板(15)螺旋固定,过滤板(15)为20mm×厚18mm的钢套筒在中间卡住若干层不锈钢网,目的是在进行粒子轰击时微粒子从阻挡板(13)喇叭口射出而进入样品室(16)中的动、植物细胞内,不锈钢网则把一些小碎片再进行一次过滤作用,枪管(11)上和阻挡板(13)上均有出气孔,其作用是为了泄漏加速后大型射弹在枪管中形成的压缩空气的冲击波,另一作用是在粒子轰击系统用钢室密封抽真空时,它们又是抽气孔,阻挡板(13)开有孔经φ1.2mm-0.9mm×深度0.6mm-1.0mm×锥角50°-70°的喇叭发射孔(14)。 (13) with the central barrier plate made of hardened steel horn opened emission hole (14), and that the filter plate (15) screwed filter plate (15) of 20mm × 18mm thick steel sleeve intermediate card several layers of stainless steel mesh live, fine particles from the object blocking plate (13) is emitted into the sample chamber of the bell mouth (16) during the moving particle bombardment, plant cells, a number of small pieces of stainless steel mesh put once again filtration the barrel (11) has an upper and a blocking plate (13) holes, whose role is to leak the compressed air to accelerate the large projectile shockwave formed in the barrel, the other function is particle bombardment systems in steel when the sealed chamber is evacuated, they are also the suction holes, the blocking plate (13) is opened φ1.2mm-0.9mm × depth speaker emission hole (14) × cone angle of 50 ° -70 ° hole by 0.6mm-1.0mm. 阻挡板是用淬火钢做成的φ20mm×厚10mm钢板,阻挡板(13)的作用是做第一级阻挡引爆后的大型射弹通过,并且起到缓解射弹飞行时由于压缩枪管中的空气形成的冲击波,枪管(8)是由φ20mm×长70mm钢管做成,上面有若干椭园形出气孔可泄漏大部分冲击波。 Barrier φ20mm × 10mm thick steel plate is made of hardened steel, the blocking action plate (13) is to do a first stage large barrier detonated by the projectile, and functions to ease the compression flying projectile in the barrel air to form blast, barrel (8) is made of steel φ20mm × 70mm long, there may be a plurality of elliptical vents most of the shock wave leakage above. 在进行全封闭状态操作时,即把钢室(17)套在枪上的密封垫圈(10)上并固定住,钢室的内径决定密封垫圈(10)的大小,它们要紧密配合。 Closed state during operation, i.e. the steel chamber (17) on the gun sleeve gasket (10) and fixed, determines the size of the inner diameter of the steel chamber sealing gasket (10), which are to mate. 在套上钢室前先把样品室(18)内装好要轰击的离体动、植物样品,样品放在一个金属培养皿中心,培养皿大小刚好固定在样品室内固定底座上,这样以实现轰击时的定位准确。 Good built to bombardment prior to body motion from the steel sleeve first chamber of the sample chamber (18), a plant sample, a sample was placed in a metal center of the dish, just the size of the dish is fixed to the fixed sample chamber base, to achieve such bombardment positioning accuracy. 然后把样品室(18)固定在过滤板(15)上,再把钢室(17)的抽气嘴(19)与真空泵接好,即可对粒子枪的轰击系统和枪体部分抽真空了,当在进行对动、植物的原体原位进行轰击时不需在封闭状态下工作,即可不用套上钢室和样品室,而是把一个消音套筒(20)套在密封垫圈(10)上,消音套筒如图四所示,它的内径大小只要与密封圈(10)刚好紧密配合为准,用卡子把它们固定住,一般采用金属做成,它的作用是为了在操作时保护操作人员的安全并把冲击波缓解在保护筒内。 Then the sample chamber (18) is fixed to the filter plate (15), and then the steel chamber (17) of the suction nozzle (19) and a vacuum pump is connected, and the system can bombardment gun body portion of the particle gun is evacuated without performing the work when moving to the original plants were bombarded in situ in the closed state, you can not put the steel chamber and the sample chamber, but to a silencer sleeve (20) in the sleeve gasket ( 10), the muffler of the sleeve shown in Figure IV, so long as the size of its internal diameter and the seal ring (10) subject to a tight fit exactly, with the clamp them fixed, generally made of metal, its role is to operate in when security to protect the operator and ease the shock wave in a protective cartridge. 操作时操作人员可手握住消音保护筒(20),对准原体原位动、植物样品进行轰击。 When the operator may operate the hand holding the muffler protective cylinder (20), aligned with the former in situ animal and plant samples were bombarded.

实施例1:用杨州生产的射针枪做转移枪的主体,轰击速度为640米/秒,火药弹配比为100%的通常火药做成6.35mm大小的大型射弹为φ7.8mm×长20mm的尼龙弹,用钢做成的内径φ40mm的密封塞(9)套在枪口上,密封塞外套上φ50mm的橡皮密封圈(10),密封塞(9)的另一端面内径开有一个洞与φ20mm×长70mm钢管做的枪管(11)紧密配合插入,并在枪管(11)上开有三个椭园形出气孔(12),枪管(11)的另一端与内径为φ20mm×厚10mm淬火钢做成的连接头(16)螺旋配合固定,连接头(16)上面周围均匀开有φ4mm的8个出气孔。 Example 1: Yang states produced by the body to make the transfer needle gun shot gun bombardment speed of 640 m / s, powder ratio of 100% elastic powder made generally large size of 6.35mm projectile is φ7.8mm × 20mm long nylon shells, made of steel of the inner diameter of φ40mm sealing plug (9) at the muzzle sleeve, the sealing sleeve φ50mm rubber stopper outer ring (10), the other end face of the inner diameter of the sealing plug (9) has an opening φ20mm × 70mm long holes and barrel (11) is inserted into the steel pipe to make a tight fit, and on the barrel (11) is opened three oblong vent (12), the barrel (11) and the other end of the inner diameter of Ø 20 mm × 10mm thick made of hardened steel connecting head (16) with a spiral fixed connector (16) above ambient uniformly opened φ4mm eight vent. 阻挡板(13)具有一个φ1mm、深度为0.8mm、锥角为60°的喇叭型发射孔,淬火钢做的,与联接头(16)相接。 Blocking plate (13) having a diameter of 1 mm, a depth of 0.8mm, 60 ° cone angle of the horn-type emitter hole, made of hardened steel, with the coupling head (16) in contact. 过滤板(15)底部固定有三层不锈钢网,网的大小正好卡在过滤板(15)内径里,底部样品室(18)用60m×长55mm的钢做成,它的一端与过滤板螺旋相配合,另一端在装好样品后用φ50螺纹板封盖。 (15) fixed to the bottom of the filter plate with three layers of stainless steel mesh, the mesh size of the inner diameter just stuck in the filter plate (15), the bottom of the sample chamber (18) made of steel with a 60m × 55mm long, one end of the filter plate with a helical with, at the other end installed with a sample φ50 threaded plate closure. 钢室(17)为φ150mm×240mm×壁厚5mm上有抽气孔(19)接机械泵,机械泵型号:2HZ-I型旋片式真空泵,真空表用自动化仪表三厂生产的。 Steel chamber (17) of the φ150mm × 240mm × 5mm thickness with a suction hole (19) connected to a mechanical pump, mechanical pump type: 2HZ-I type rotary vane vacuum pump, vacuum gauge automated production Instrument Factory.

实施例2:在半封闭状态下操作用的粒子枪,枪体(一)、轰击系统(二)均与实施例1相同,在实施例1中取下钢室(17)和样品室(18),在密封垫圈(10)上套上φ52mm×长110mm×厚6mm的铝合金做的消音保护筒(20),用铁卡固定住,火药弹用φ6.35mm大小的一个,火药配比为70%的通用火药,30%的碳粉做填充剂。 Example 2: Operation with a particle gun, the gun body (a), bombardment system (II) are the same as in Example 1 in a semi-closed state, the steel is removed in embodiment 1 Embodiment chamber (17) and the sample chamber (18 ), sleeve length 6mm aluminum φ52mm × 110mm × thickness do muffler protective cylinder (20) on the sealing gasket (10), fixed with iron card, the size of the 6.35 mm projectile with a powder, ratio of gunpowder 70% of the common gunpowder, do 30% of toner filler. 实现火药速度为460米/秒。 Implemented gunpowder speed 460 m / sec.

本发明提供的一种转移基因的物理方法简便易行,进行轰击粒子的速度是根据火药弹的配比来控制实现的,这种方法定量准确,速度控制准确,可予先根据被射入基因的动、植物细胞壁厚、薄及坚硬程度来决定选择轰击的速度即可选择火药的配比,这样避免了由于轰击速度不当而造成外源基因射不进去或者使受体的细胞遭到过量冲击导致细胞不必要的损失,以至于把细胞打坏。 Physical method of transferring a gene of the present invention provides a simple, particle bombardment speed is controlled according to the ratio of powder to achieve the projectile, this method is accurate, precise speed control, according to the first incident may be the gene animal and plant cell wall thickness, a thin and hard to determine the degree to select the selected velocity bombardment gunpowder ratio, thus avoiding excessive impact velocity bombardment caused due to improper exogenous gene is not emitted into the cell or the receptor was resulting in unnecessary loss of cells, so the cells broke.

本发明提供的专用设备转移基因粒子枪设计合理、小巧,不需要把整个枪密封在真空系统中,只需在枪口上固定一个粒子轰击系统,这部分满足了在封闭状态下进行轰击操作,改变了已有技术不足地方,另外这部分也可以不封闭,开创了可在大气中对生物原体原位进行外源基因的导入特殊功能,这是已有技术所不具备的,将为生物工程中实现各种生物的外源基因转移技术提供了极为有力的工具。 Reasonable gene transfer particle gun equipment according to the present invention provides design, compact, does not require the entire gun sealed in a vacuum system, only a particle bombardment system fixed on the muzzle, which satisfies the bombarded part of the operation in the closed state, change the lack of prior art where, in addition to this section may not be closed, creating a special import function allows foreign gene to the original biological in situ in the atmosphere, which is not available in the prior art, for Bioengineering implemented in various biological exogenous gene transfer technology provides a very powerful tool.

Claims (7)

1.一种采用粒子枪轰击转移基因的方法,其特征在于:在粒子枪的火药弹前面载有大型射弹,射弹的前表面载有微钨粒子,微钨粒子上带有外源基因,通过火药的发射轰击把带有外源基因的微粒子加速到受体之内,其轰击速度由火药配比决定关系:采用640米/秒轰击速度用φ6.35mm火药弹内装100%的火药,460米/秒70%-90%火药和30%-10%的填充剂,350米/秒的轰击速度火药配比为60%-80%火药和40%-20%的填充剂,轰击时可在封闭状况下0.1大气压以上进行;也可以在大气中操作。 CLAIMS 1. A method of gene transfer particle gun bombardment, characterized in that: the projectile carrying a large particle gun in front of the powder shells, a front surface of the projectile carrying the micro tungsten particles with micro foreign gene tungsten particles , the bombardment with fine particles of foreign genes into the receptor accelerated by the propellant, which bombardment rate determined by the relationship between the ratio of powder: a 640 m / s velocity bombardment with gunpowder shells built φ6.35mm 100% powder, 460 m / s and 70% -90% 30% -10% powder filler 350 m / s velocity bombardment powder ratio of 60% -80% 40% -20% and the powder filler may bombardment for more than 0.1 atm at a closed condition; it may be performed in air.
2.按权利要求1所述的转移基因的方法,其特征在于:所说的火药为市售的通用火药,火药填充剂包括碳粉及通用的火药填充剂。 2. A method for transferring a gene according to claim 1, wherein: said powder is powder commercially available general, powder fillers include carbon powder filler and general gunpowder.
3.按权利要求1所述的转移基因的方法,其特征在于:所说的微钨粒子的直径范围从0.6μm-4μm几个量度。 3. The method for transferring a gene according to claim 1, wherein: the diameter of said micro-tungsten particles range from 0.6μm-4μm several metrics.
4.按权利要求1所述的转移基因的方法,其特征在于:所说的大型射弹可以是一般塑料可以是尼龙做成。 4. A method for transferring a gene according to claim 1, wherein: said large projectile may be generally made of plastic may be nylon.
5.一种专用于权利要求1所述的转移基因的方法的转移基因粒子枪,由枪把(1)、板机(2)、安全钮(3)、击针(4)、火药弹(5)、枪管(8)组成的,其特征在于:在枪口处固定一个套有橡皮密封垫圈(10)的金属密封塞(9),密封塞(9)另一端固定在带有出气孔(12)的枪管(11)上,枪管(11)的另一端与金属连接头(16)上开有出气孔的一端相联,用淬火钢板做的阻挡板(13)中央有一个喇叭型发射孔(14),它与联接头(16)螺旋固定,另一端与带若干层的不锈钢网过滤板(15)螺旋相接,火药弹(5)装在枪管(8)内固定座上,大型射弹(6)放在火药弹(5)的前表面上,大型射弹(6)前表面载有带外源基因的微钨粒子,然后把金属消音保护筒(20)套在密封垫圈(10)上;或者取下消音保护筒(20),样品室(18)固定在过滤板(15)上再套 A specific method of gene transfer particle gun in transferring a gene according to claim 1, a gun (1), the trigger (2), the safety knob (3), firing pin (4), gunpowder shells ( 5), the barrel (8) composition, characterized in that: a sleeve fixed to the metal with a rubber sealing gasket (10) sealing plug (9) at the muzzle, the sealing plug (9) at the other end with a vent the barrel (11) (12), opened on the other end of the barrel (11) with a metal connector (16) at one end of the associated hole, made of hardened steel barrier plate (13) has a central speaker type emitter hole (14), with which the coupling head (16) screwed, with the other end of the stainless steel mesh filter plate with a plurality of layers (15) in contact with the coil, gunpowder shells (5) mounted within the barrel (8) holder , the large projectile (6) on the front surface of the powder shells (5), and a large projectile (6) carrying the front surface of the tungsten particles with micro foreign gene, then the cancellation metal protective cylinder (20) in the sleeve a sealing gasket (10); removing the muffler or the protective tube (20), the sample chamber (18) is fixed on the filter plate (15) and then set 钢室(17)在密封垫圈(10)上,真空泵按到钢室(17)的抽气孔(21)上。 Steel chamber (17) on the sealing gasket (10), the steel chamber by a vacuum pump (17) of the suction holes (21).
6.按权利要求5所述的转移基因的粒子枪,其特征在于:所说的金属消音保护筒(20)可以是铝、铝合金。 6. The particle gun according to claim 5, wherein the gene transfer, wherein: said metal muffler protective cylinder (20) may be aluminum, an aluminum alloy.
7.按权利要求5所述的转移基因的粒子枪,其特征在于:所说的带不锈钢网的过滤板(15)至少含有二层以上的不锈钢网。 7. The particle gun according to claim 5, wherein the gene transfer, wherein: said strip of stainless steel mesh filter plate (15) comprises at least two or more layers of stainless steel mesh.
CN 89109334 1989-12-22 1989-12-22 Transfer genes method and particle gun CN1052695A (en)

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