CN1058291C - Cell modification technique with low energy ion beam and device - Google Patents

Cell modification technique with low energy ion beam and device Download PDF

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CN1058291C
CN1058291C CN 93103361 CN93103361A CN1058291C CN 1058291 C CN1058291 C CN 1058291C CN 93103361 CN93103361 CN 93103361 CN 93103361 A CN93103361 A CN 93103361A CN 1058291 C CN1058291 C CN 1058291C
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energy
modification
beam
technique
ion
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CN 93103361
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CN1077495A (en )
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余增亮
何建军
杨剑波
吴跃进
陈备久
周骏
沈玉琴
孙洪奎
尹载群
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中国科学院等离子体物理研究所
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/02Electrical or electromagnetic means, e.g. for electroporation or for cell fusion

Abstract

本发明是低能离子束细胞修饰技术和装置。 The present invention is a low energy ion beam Cell modification techniques and devices. 本发明技术是用低能离子束溅射刻蚀对放在微环境靶室内的并经过预冷的动、植物细胞进行溅射刻蚀,外源基因导入,细胞融合等。 The techniques of this invention with a low energy ion-beam sputter etching on the micro-environment of the target chamber and moving through the pre-chilled, sputter etching plant cell, introducing an exogenous gene, cell fusion. 本发明装置是由一个离子源,一个可使离子束通过的主真空室和一个微环境靶室组成。 The present invention is an ion source means, a primary ion beam enables the vacuum chamber and the target chamber through a microenvironment of the composition. 微环境靶室内装有样品架,配有预抽系统,装有高效过滤材料和气源接口的气流净化结构。 Microenvironment target chamber containing the sample holder, with a pre-pumping system, equipped with high efficiency filter material and gas purge gas source structure of the interface. 主真空室与微环境靶室是由隔离阀连接的。 And the main vacuum chamber microenvironment target chamber isolation valve is connected.

Description

低能离子束细胞修饰技术和装置 Cell modification techniques low energy ion beam apparatus and

本发明涉及生物工程,国际分类号C12N 13/00;G01N1/00。 The present invention relates to biotechnology, International Classification C12N 13/00; G01N1 / 00.

在植物细胞工程中,操作的受体为原生质体,但是由于纤维素酶,果胶酶等的酶解作用和较长时间的去壁操作,使细胞受到一定的损害。 In engineering a plant cell, the receptor for the operation protoplasts, but due to enzymatic hydrolysis wall cellulase, pectinase and longer operating time, subject to certain damage to the cells. 加之,不少植物,特别是禾谷类作物的原生质体再生植株相当困难,因此在一定程度上限制了这些技术的应用。 Additionally, many plants, particularly cereals protoplasts regenerated plants very difficult, thus limiting the application of these techniques to a certain extent. 曾经有不少人采用物理方法,但因各自都有不可避免的缺点,如:电击法、超声波法、γ射线等电离辐射方法,其主要受体仍为原生质体;基因枪法,则由于金属粉载体的毒害作用,效果也不很理想;激光法,属于热蒸发一类,虽可在细胞壁上打孔,但极易伤及临近的细胞器,加之是单个细胞操作,效率低。 Many people who have physical method, but each has unavoidable disadvantages, such as: electric shock, ultrasound, ionizing radiation such as gamma] ray method, protoplast still major receptor; gene gun, since the metal powder toxicity of the carrier, the effect is not very satisfactory; laser method, a thermal evaporation belonging to a class, although in the cell wall can be perforated, but easily hurt adjacent cells, a single cell is combined with the operation inefficient.

本发明的第一个目的就是提供一种采用低能离子束对细胞进行修饰从而达到改良物种的方法;本发明的另一个目的,就是提供一种实现低能离子束对细胞进行修饰的装置。 A first object of the present invention is to provide a low energy ion beam method to achieve the modified cells are modified species; a further object of the present invention, there is provided a means to achieve low energy ion beam of modified cells.

采用离子注入作物诱变育种技术,对于促进农作物改良起了重要作用。 Ion implantation Mutagenisis crop, crop improvement for promoting play an important role.

离子注入作物诱变育种的原理是:将离子注入到生物体某一薄层内,通过注入离子与生物分子的能量交换和慢化离子的掺杂,引起这一薄层内的基因突变和重组,从而达到改良物种的目的。 Ion implantation mutation breeding crops principle is: implanting ions into a thin layer within a living body, by the implantation energy of ion exchange biomolecule and moderator ions doped, and gene mutation causing recombination within this thin layer , so as to achieve improved species.

由于离子的射程是可控的,因而对种子的损伤是局部的,而在局部区域的诱变作用又是双重的:即能量交换和质量沉积。 Since the range of the ions is controlled, and thus damage to the seed is local, and the local region mutagenesis is twofold: the mass and energy exchange deposition. 因此,在物种成活率高的情况下可获得高的突变率和宽的突变谱据联合国粮农组织统计,γ射线诱变在半致死剂量下,平均突变率为1%,有利突变率万分之三;而离子注入诱变在成活率90%的情况下,平均突变率8.4%,有利突变率1%。 Thus, at high survival rate of the species the availability of a high mutation rate and a wide spectrum of mutations in the FAO, according to statistics, gamma] rays irradiation in the half lethal dose, average mutation rate of 1%, extremely advantageous mutation rate of three; and in the case of ion implantation mutagenesis survival rate of 90%, the average mutation rate of 8.4%, advantageously 1% mutation rate. 由此可见,离子注入诱变育种的效率比γ射线高几十倍甚至上百倍。 Thus, the efficiency of the ion implantation mutation breeding several times higher than that of γ-rays or even a hundred times. 更重要的是,由于离子束与生物体相互作用在入射方向上有高度的空间分辨率,如果离子束聚焦到微米束以下,就可以实现三维空间分辨。 More importantly, due to the interaction of the ion beam with a high degree of biometric spatial resolution in the incident direction, if the ion beam is focused to a beam microns or less, a three-dimensional spatial resolution can be achieved. 人们可以利用这一技术进行基因位点操作,从而解决诱变育种的方向性问题。 People can use this technology to gene loci operation, so as to solve the problem directional mutation breeding.

本发明是利用低能离子束溅射刻蚀细胞壁,使之在细胞壁上形成微孔,离子通过微孔作用在细胞膜上,形成生物大分子(如外源基因分子)进入细胞的通道。 The present invention utilizes low energy ion beam sputter etching cell walls so as to form pores in the cell wall, acting on the ions through the porous membrane is formed of biological macromolecules (e.g., a foreign gene molecule) into a cell passage.

本发明是借助低能离子与细胞表面动量交换原理,适应各种动、植物细胞诱变、去壁的细胞修饰技术。 The present invention is a cell surface by means of low energy ions and the momentum transfer principle, adapt to a variety of animal and plant cells mutagenesis, to cell wall modification techniques.

本发明的方法,是在无菌条件下,将动植物细胞以0.5度/分的速度使之预冷到4-6度,通过无菌室,放入微环境靶室,靶室的真空度为2×10-4~2×10-6乇。 The method of the present invention, under sterile conditions, so that the animal and plant cells at a rate of 0.5 / min of pre-cooled to 4-6 degrees, through the sterile room, placed in the microenvironment of the target chamber, the target degree of vacuum chamber of 2 × 10-4 ~ 2 × 10-6 Torr.

本发明还需根据不同的目的,选择不同的离子:作细胞诱变时,注入N+,能量为30-50KeV,剂量在1×1015~1×1016之间;作细胞壁刻蚀时,选择Ar+,能量为10~20KeV,剂量在1×1014~1×1015之间。 The present invention is needed for different purposes, different ion selected: when making somatic mutagenesis, implanted N +, energy 30-50KeV, doses between 1 × 1015 ~ 1 × 1016; etching time for the cell wall, selected Ar +, energy of 10 ~ 20KeV, doses between 1 × 1014 ~ 1 × 1015.

当两细胞靠得很近时,用Ar+离子束来扫过,进行细胞融合;当壁被离子束刻蚀后,进行外源基因导入。 When two cells in close proximity, by Ar + ion beam sweeps the cell fusion; wall when ion beam etching is performed exogenous gene.

根据热力学原理,汽液相面的平衡湿度与蒸汽压有关。 The thermodynamic principles, the vapor phase equilibrium moisture vapor pressure and surface-related. 当气压下降时,相面温度会随之而下降。 When the pressure drops, physiognomy temperature will follow the decline. 假设一个细胞为一个半径为r的小水滴,当它瞬间暴露在10-2乇以下的真空时,细胞表面一层将迅速结冰,形成厚度为dr的冰壳。 Suppose a cell is a small drop of radius r, and the moment when it is exposed to the vacuum of 10-2 Torr or less, a cell surface layer will rapidly freeze, forming ice shell thickness dr. 随着时间的推移,冰壳向内部延伸。 Over time, extend into the interior of the ice shell. 冰壳的存在防止内部水份的蒸发,并有足够的强度承受负压,使细胞不致破坏。 The presence of ice inside the housing to prevent evaporation of moisture, and have sufficient strength to withstand the negative pressure, will not damage the cells. 但当细胞放置在真空室内的时间过长,细胞表层的冰壳将会因承受不住真空室里的负压而破裂。 However, when cells were placed in a vacuum chamber for too long, the cell surface layer by the ice shell will withstand the negative pressure of the vacuum chamber is broken. 为此,本发明设计了低能离子束细胞修饰技术的装置。 To this end, the present invention contemplates low energy ion beam apparatus of cell modification techniques.

本发明装置包括:一个离子源,一个主真空室,该真空室配有真空机组,留有一通道,可以让离子源引出的离子束通过,注入到放在样品架上的动植物细胞上。 The present invention apparatus comprising: an ion source, a main vacuum chamber, the vacuum chamber equipped with a vacuum unit, a left channel, so that the ion beam can be drawn by the ion source, injected onto the sample holder in animal and plant cells.

一个小真空室,亦称微环境靶室,该靶室是通过隔离阀与大真空室相连,靶室内有用于放置动、植物细胞物种的样品架,靶室配一个预抽泵,还有一个装有高效过滤材料的气流净化结构和一个气源接口。 A small vacuum chamber, known as the micro-environment of the target chamber, through the target chamber isolation valve and is connected to a large vacuum chamber, for placing the movable target chamber, the species of plant cell sample holder, the target chamber with a pre-pumped, there is a an air-flow and a purge air supply connection structure of high efficiency filter material.

工作时,首先将大真空室抽至2×10-6,将离子源调试正常,在无菌的环境下,把待处理的动植物细胞放在小真空靶室的样品架上,再把小真空室相连的预抽泵打开,抽真空1分钟,使小真空室的真空度达10-2乇左右,打开隔离阀,小真空室内的真室度迅速达到2×10-6乇,同时,离子源放电引出,荷能离子注入到样品架上,即对动、植物细胞进行刻蚀、打孔、去核,以及外源基因转移,细胞融合等。 In operation, a large vacuum chamber is first evacuated to 2 × 10-6, debug the normal ion source, in a sterile environment, plant and animal cells to be treated placed on the sample holder target small vacuum chamber, and then a small pre-evacuating the vacuum chamber connected to the pump is opened, evacuated for 1 minute to a small degree of vacuum of the vacuum chamber was about 10-2 Torr, the isolation valve is opened, a small chamber of the vacuum chamber quickly to the true 2 × 10-6 Torr, at the same time, discharge ion source extraction, energetic ions are implanted into the sample holder, i.e. of the animal and plant cells etching, punching, to the core, and an exogenous gene transfer, cell fusion.

本发明装置的特点在于,有一个小真空室,而大小真空室的体积比为1000∶2,小真空室的真空度只需预抽到10-1~10-2乇,打开阀,小真空室就可在极短的时间内真空度达到10-8左右,这样,放在样品架上的细胞,只需在小真空室内放置10分钟以内,就可完成离子注入,从而保护了被处理的细胞。 Feature of the present invention means that there is a small vacuum chamber, the volume ratio of the size of the vacuum chamber is 1000:2, a small degree of vacuum of the vacuum chamber of 10-1 to 10-2 drawn only pre Torr, the valve is opened, a small vacuum chamber can be in a very short period of time to achieve a degree of vacuum of about 10-8 so that the sample rack on the cell, only a small vacuum chamber is placed within 10 minutes, and ion implantation can be completed, thereby protecting the treated cell.

试验表明:对棉花花粉母细胞,在小真空室放置4小时,成活率为20.1%,而放置25分钟,成活率为96.0%,这相当于20分钟内40℃大气条件下的存活率(94.7%)。 Test showed that: cotton pollen mother cells, is placed in a small vacuum chamber 4 hours, the survival rate was 20.1%, and placed for 25 minutes, the survival rate was 96.0%, which is equivalent to the survival rate at 40 ℃ atmospheric conditions over 20 minutes (94.7 %).

本发明装置使得离子刻蚀过程中(10分钟内)排除了真空对细胞成活率的影响。 Apparatus of the present invention is such that the ion etching process (over 10 min) to eliminate the influence of vacuum to the cell viability.

本发明装置的效果是好的。 Effect of the present invention apparatus is good.

1.动植物细胞在靶室内滞留时间10分钟,承受5×1016个离子/平方厘米刻蚀后,存活率对各种单细胞生物可达30-70%。 1. The residence time in the animal and plant cells target chamber for 10 minutes and subjected to 5 × 1016 ions / cm etching, a variety of unicellular organisms survival up to 30-70%.

2.GUS CAT和PBI222报导基因经离子束介导转入水稻完整细胞和成熟胚获高水平表达,并获大量的转基因植株。 PBI222 2.GUS CAT reporter gene, and by ion beam-mediated transformation of rice mature embryos obtained intact cells and high level expression, and a large number of transgenic plants obtained.

3.棉花花粉细胞和酵母菌的离子束细胞融合。 3. Cotton pollen cells and yeast cell fusion ion beam.

4.洋葱培养细胞的去核。 4. onion enucleated cell culture.

5.带有标记基因2.7Kb的质粒导入大肠杆菌,离子注入体内质粒,突变率30%。 The 2.7Kb plasmid bearing the marker gene introduced into E. coli, the ion implantation in vivo plasmid, the mutation rate of 30%.

附图说明 BRIEF DESCRIPTION

:1 离子源 2 主真空室3、9 样品架 4 隔离阀5 汽流净化结构 6 过滤7 气源接口 8 预抽接口10 冷却 11 微环境靶室12 真空机械泵 13、14 真空机组 : A main vacuum chamber of the ion source 2 sample holder 4 3,9 5 bleed isolation valve structure purifying filter 6 7 8 pre-evacuating air interface 10. Interface 11 microenvironment target cooling chamber 12 vacuum unit 13, vacuum mechanical pump

Claims (5)

  1. 1.一种低能离子束细胞修饰技术,其特征是用低能离子束对放在微环境靶室内的,并经过预冷过的动、植物细胞进行注入、溅射和刻蚀,所述的离子束,在作细胞诱变时注入N+离子,能量为30~50KeV,剂量为1×1016~1×1016,在作细胞壁刻蚀时用Ar+离子,能量为10~20KeV,剂量为1×1014~1×1015,所述的微环境靶室内的真空度为2×10-4~2×10-6乇。 A low energy ion beam Cell modification techniques, wherein the low-energy ion beam in the microenvironment of the target chamber, and movable through a pre-cooled, plant cell implantation, sputtering and etching, the ion beam injected in making somatic mutagenesis N + ions with an energy of 30 ~ 50KeV, a dose of 1 × 1016 ~ 1 × 1016, using Ar + ions in making the cell wall etching, an energy of 10 ~ 20KeV, a dose of 1 × 1014 ~ 1 × 1015, the micro-environment of the target degree of vacuum chamber was 2 × 10-4 ~ 2 × 10-6 Torr.
  2. 2.根据权利要求1所述的技术,其特征是动、植物细胞在离子注入、溅射和刻蚀前,是以0.5度/分的速度预冷到4-6度。 2. The technique according to claim 1, characterized in that the movable, plant cell ion implantation, sputtering and etching before, at 0.5 degree / min for precooled to 4-6 degrees.
  3. 3.根据权利要求1所述的技术,其特征是当两细胞靠得很近时,用Ar+离子束来扫过,进行细胞融合;当细胞壁被离子刻蚀后,进行外源基因导入。 3. The technique according to claim 1, characterized in that when the two cells are close together, with the Ar + ion beam sweeps the cell fusion; ions when the cell walls are etched, for exogenous gene.
  4. 4.一种低能离子束细胞修饰装置,其特征是配接离子源(1)和真空机组(13,14)的主真空室(2)内置有样品架(3),所述的主真空室(2)经隔离阀(4)与内设样品架(9)的微环境靶室(11)相连接,所述的微环境靶室(11)配接有气源接口(7)和预抽接口(8)。 A low energy ion beam apparatus modified cells, characterized in that the main vacuum chamber is connected with an ion source (1) and the vacuum unit (13, 14) (2) built sample holder (3), said main vacuum chamber (2) is connected via an isolation valve (4) equipped with a sample holder (9) of the micro-environment of the target chamber (11), the micro-environment of the target chamber (11) connected with gas source interface (7) and the pre-extraction an interface (8).
  5. 5.根据权利要求4所述的装置,其特征是气源接口(7)与微环境靶室(11)间串接有气流净化结构(5)和过滤材料(6)。 5. The apparatus according to claim 4, characterized in that the air supply connection connected in series with a purge gas flow structure (5) and a filter material (6) between (7) and the micro-environment of the target chamber (11).
CN 93103361 1993-03-26 1993-03-26 Cell modification technique with low energy ion beam and device CN1058291C (en)

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CN106531604A (en) * 2016-10-27 2017-03-22 合肥优亿科机电科技有限公司 Biological modification equipment employing high-density and low-energy ion beams of hot cathode

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN86108459A (en) * 1985-12-11 1987-07-29 株式会社岛津制作所 Apparatus for fusing cells
CN86103459A (en) * 1985-04-18 1987-11-18 澳洲生物科技公司 Recombinant inhibin
CN1057482A (en) * 1991-07-04 1992-01-01 北京理工大学 Electricity transferring device for cell electric fusion

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN86103459A (en) * 1985-04-18 1987-11-18 澳洲生物科技公司 Recombinant inhibin
CN86108459A (en) * 1985-12-11 1987-07-29 株式会社岛津制作所 Apparatus for fusing cells
CN1057482A (en) * 1991-07-04 1992-01-01 北京理工大学 Electricity transferring device for cell electric fusion

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