CN1058291C - Cell modification technique with low energy ion beam - Google Patents
Cell modification technique with low energy ion beam Download PDFInfo
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- CN1058291C CN1058291C CN93103361A CN93103361A CN1058291C CN 1058291 C CN1058291 C CN 1058291C CN 93103361 A CN93103361 A CN 93103361A CN 93103361 A CN93103361 A CN 93103361A CN 1058291 C CN1058291 C CN 1058291C
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- cell
- ion beam
- target chamber
- ion
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- 238000010884 ion-beam technique Methods 0.000 title claims abstract description 14
- 230000004048 modification Effects 0.000 title claims abstract description 6
- 238000012986 modification Methods 0.000 title claims abstract description 6
- 238000000034 method Methods 0.000 title claims description 13
- 238000005530 etching Methods 0.000 claims abstract description 9
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 8
- 238000005516 engineering process Methods 0.000 claims abstract description 6
- 239000000463 material Substances 0.000 claims abstract description 3
- 210000004027 cell Anatomy 0.000 claims description 37
- 230000035772 mutation Effects 0.000 claims description 12
- 238000005468 ion implantation Methods 0.000 claims description 8
- 210000002421 cell wall Anatomy 0.000 claims description 6
- 239000012530 fluid Substances 0.000 claims description 6
- 230000024835 cytogamy Effects 0.000 claims description 4
- 230000008676 import Effects 0.000 claims description 3
- 238000000992 sputter etching Methods 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims 1
- 230000007910 cell fusion Effects 0.000 abstract 1
- 238000000605 extraction Methods 0.000 abstract 1
- 238000001659 ion-beam spectroscopy Methods 0.000 abstract 1
- 238000002955 isolation Methods 0.000 abstract 1
- 238000000746 purification Methods 0.000 abstract 1
- 238000004544 sputter deposition Methods 0.000 abstract 1
- 241000196324 Embryophyta Species 0.000 description 11
- 150000002500 ions Chemical class 0.000 description 11
- 238000002703 mutagenesis Methods 0.000 description 5
- 231100000350 mutagenesis Toxicity 0.000 description 5
- 241000894007 species Species 0.000 description 4
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 229920000742 Cotton Polymers 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000002349 favourable effect Effects 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 244000291564 Allium cepa Species 0.000 description 1
- 235000002732 Allium cepa var. cepa Nutrition 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 108010059820 Polygalacturonase Proteins 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 235000019628 coolness Nutrition 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000000151 deposition Methods 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000005251 gamma ray Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 231100000636 lethal dose Toxicity 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000002220 organoid Anatomy 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 238000004080 punching Methods 0.000 description 1
- 230000008521 reorganization Effects 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000002344 surface layer Substances 0.000 description 1
- 238000002207 thermal evaporation Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS 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
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Sustainable Development (AREA)
- Physics & Mathematics (AREA)
- Biomedical Technology (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Electromagnetism (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
本发明是低能离子束细胞修饰技术和装置。
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。
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。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。主真空室与微环境靶室是由隔离阀连接的。
The invention is a low-energy ion beam cell modification technology and device.
The technology of the present invention uses low-energy ion beam sputtering etching to carry out sputtering etching on precooled animal and plant cells placed in a microenvironmental target chamber, exogenous gene introduction, cell fusion and the like.
The device of the invention is composed of an ion source, a main vacuum chamber through which the ion beam can pass and a microenvironment target chamber. The micro-environment target chamber is equipped with a sample rack, a pre-extraction system, and an airflow purification structure equipped with high-efficiency filter materials and air source interfaces. The main vacuum chamber and the microenvironment target chamber are connected by an isolation valve.
Description
The present invention relates to biotechnology, international classification number C12N 13/00; G01N1/00.
In plant cell engineering, the acceptor of operation is a protoplastis, but because cellulase, the wall that goes of the enzymolysis of polygalacturonase etc. and long period is operated, and makes cell be subjected to certain infringement.In addition, the protoplast regenerated plant of many plants, particularly cereal crop is difficulty quite, has therefore limited these The Application of Technology to a certain extent.Once there were many people to adopt physical method, but because of inevitable shortcoming is all arranged separately, as: ionizing rays methods such as electric shocking method, supersonic method, gamma-rays, its principal recipient still is a protoplastis; Particle bombardment, then owing to the toxic action of metal powder carrier, effect is also not really desirable; Laser method belongs to thermal evaporation one class, though can punch on cell walls, very easily injures the organoid that closes on, and is the individual cells operation in addition, and efficient is low.
Thereby first purpose of the present invention just provides a kind of method that adopts the low energy ion beam pair cell to modify to reach the improvement species;
Another object of the present invention just provides a kind of device of realizing that the low energy ion beam pair cell is modified.
Adopt ion implantation mutagenesis crops breeding technique, for promoting the farm crop improvement to play vital role.
The principle of ion implantation mutagenesis crops breeding is: arrive in the organism skim ion implantation, mix by energy exchange and the slowing down ionic that injects ion and biomolecules, cause transgenation and reorganization in this skim, thereby reach the purpose of improvement species.
Because the ionic range is controlled, thereby be partial, and be dual: be i.e. energy exchange and quality deposition at the mutagenesis of regional area to the damage of seed.Therefore, under the high situation of species surviving rate, can obtain high mutation rate and wide mutation spectrum
According to Food and Argriculture OrganizationFAO's statistics, gamma-ray and mutagenesis is under medial lethal dose, and average mutation rate is 1%, favourable mutation rate 3/10000ths; And ion implantation mutagenesis is under the situation of surviving rate 90%, average mutation rate 8.4%, favourable mutation rate 1%.This shows the efficiency ratio gamma-rays of ion implantation mutation breeding high tens times even hundreds of times.The more important thing is the spatial resolution of height is arranged on incident direction, differentiate if focusing of ion beam below the micron bundle, just can be realized three-dimensional space because ionic fluid and organism interact.People can utilize this technology to carry out the gene locus operation, thereby solve the directional problems of selection by mutation.
The present invention utilizes low energy ion beam ise cell walls, makes it to form on cell walls micropore, and ion acts on the cytolemma by micropore, forms the passage that biomacromolecule (as the foreign gene molecule) enters cell.
The present invention is by low energy ion and cell surface momentum exchange principle, adapts to various animal and plant cell mutations, removes the cell modification technique of wall.
Method of the present invention is under aseptic condition, with animal and plant cells with 0.5 the degree/minute speed make it to be pre-chilled to the 4-6 degree, by sterilisable chamber, put into the microenvironment target chamber, the vacuum tightness of target chamber is 2 * 10
-4～2 * 10
-6Torr.
The present invention also needs to select different ions according to different purposes:
When making cell mutation, inject N
+, energy is 30-50KeV, dosage is 1 * 10
15～1 * 10
16Between;
When making the cell walls etching, select Ar
+, energy is 10～20KeV, dosage is 1 * 10
14～1 * 10
15Between.
When two cells lean on very closely, use Ar
+Ionic fluid comes inswept, carries out cytogamy; After wall is by ion beam etching, carries out foreign gene and import.
According to thermodynamic principles, the equilibrium moisture that vapour-liquid is practised physiognomy is relevant with vapour pressure.When air pressure descended, the phase surface temperature can descend thereupon.Suppose that a cell is that a radius is the little water droplet of r, is exposed to 10 when its moment
-2During the following vacuum of torr, cell surface one deck will freeze rapidly, and forming thickness is the nilas of dr.As time goes on, nilas extends inward.The existence of nilas prevents the evaporation of internal moisture, and has enough intensity to bear negative pressure, makes the unlikely destruction of cell.Be placed on the interior overlong time of vacuum chamber but work as cell, the nilas of cell surface layer will break because of bearing the negative pressure that does not live in the vacuum chamber.For this reason, the present invention has designed the device of cell modification technique with low energy ion beam.
Apparatus of the present invention comprise:
An ion source,
A main vacuum chamber, this vacuum chamber is furnished with the vacuum unit, leaves a passage, and the ionic fluid that can allow ion source draw passes through, and is injected on the animal and plant cells that is placed on the specimen holder.
A little vacuum chamber, also claim the microenvironment target chamber, this target chamber is to link to each other with big vacuum chamber by segregaion valve, is useful on the specimen holder of placing animal and plant cell species in the target chamber, target chamber is joined a forepump, also has an air-flow treatment facilities and the gas source interface that the high efficiency filter material is housed.
During work, at first big vacuum chamber is evacuated to 2 * 10
-6, the ion source debugging is normal, under sterile environment, pending animal and plant cells is placed on the specimen holder of little vacuum target chamber, the forepump that little vacuum chamber is linked to each other is opened again, vacuumizes 1 minute, makes the vacuum tightness of little vacuum chamber reach 10
-2About torr, open segregaion valve, the true chamber degree in the little vacuum chamber reaches 2 * 10 rapidly
-6Torr, simultaneously, ion source discharge is drawn, and energetic ion is injected on the specimen holder, promptly the animal and plant cell is carried out etching, punching, stoning, and foreign gene shifts cytogamy etc.
The characteristics of apparatus of the present invention are, a little vacuum chamber is arranged, and the volume ratio of big or small vacuum chamber are 1000: 2, and the vacuum tightness of little vacuum chamber only need be extracted into 10 in advance
-1～10
-2Torr is opened valve, and little vacuum chamber just can vacuum tightness reach 10 in the extremely short time
-8About, like this, be placed on the cell on the specimen holder, only need in little vacuum chamber, to place in 10 minutes, just can finish ion implantation, thereby protected processed cell.
Test shows: to the cotton pollen parent cell, placed 4 hours at little vacuum chamber, surviving rate is 20.1%, and places 25 minutes, and surviving rate is 96.0%, and this is equivalent in 20 minutes the survival rate (94.7%) under 40 ℃ of atmospheric condition.
Apparatus of the present invention make (in 10 minutes) in the ion etching process get rid of the influence of vacuum pair cell surviving rate.
The effect of apparatus of the present invention is good.
1. animal and plant cells 10 minutes residence times in target chamber, bear 5 * 10
16After individual ion/square centimeter etching, survival rate can reach 30-70% to various unicellular organisms.
2.GUS CAT and PBI
222Reporter gene changes the paddy rice intact cell over to and mature embryo obtains high level expression through ion-beam mediated, and obtains a large amount of transfer-gen plants.
3. cotton pollen cell and saccharomycetic ionic fluid cytogamy.
4. the stoning of onion culturing cell.
5. the plasmid that has marker gene 2.7Kb imports intestinal bacteria, plasmid in the ion implantation body, mutation rate 30%.
Description of drawings:
1 ion source, 2 main vacuum chamber
3,9 specimen holders, 4 segregaion valves
5 steam flow treatment facilities 6 filter
7 gas source interfaces 8 are taken out interface in advance
10 coolings, 11 microenvironment target chambers
12 vacuum mechanical pumps, 13,14 vacuum units
Claims (5)
1. cell modification technique with low energy ion beam is characterized in that with low energy ion beam being placed in the microenvironment target chamber, and through the animal and plant cell of precooling inject, sputter and etching, described ionic fluid injects N when making cell mutation
+Ion, energy are 30～50KeV, and dosage is 1 * 10
16～1 * 10
16, when making the cell walls etching, use Ar
+Ion, energy are 10～20KeV, and dosage is 1 * 10
14～1 * 10
15, the vacuum tightness in the described microenvironment target chamber is 2 * 10
-4～2 * 10
-6Torr.
2. technology according to claim 1 is characterized in that the animal and plant cell before ion implantation, sputter and etching, be with 0.5 degree/minute speed be pre-chilled to the 4-6 degree.
3. technology according to claim 1 is characterized in that using Ar when two cells lean on very closely
+Ionic fluid comes inswept, carries out cytogamy; After cell walls is by ion etching, carries out foreign gene and import.
4. low energy ion beam cell decorating device, it is characterized in that connecting ion source (1) and vacuum unit (13,14) main vacuum chamber (2) is built-in with specimen holder (3), described main vacuum chamber (2) through segregaion valve (4) with in establish specimen holder (9) microenvironment target chamber (11) be connected, described microenvironment target chamber (11) has connected gas source interface (7) and has taken out interface (8) in advance.
5. device according to claim 4 is characterized in that being serially connected with between gas source interface (7) and microenvironment target chamber (11) air-flow treatment facilities (5) and filtering material (6).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN93103361A CN1058291C (en) | 1993-03-26 | 1993-03-26 | Cell modification technique with low energy ion beam |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN93103361A CN1058291C (en) | 1993-03-26 | 1993-03-26 | Cell modification technique with low energy ion beam |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1077495A CN1077495A (en) | 1993-10-20 |
| CN1058291C true CN1058291C (en) | 2000-11-08 |
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ID=4984612
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN93103361A Expired - Fee Related CN1058291C (en) | 1993-03-26 | 1993-03-26 | Cell modification technique with low energy ion beam |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1058291C (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN110295351B (en) * | 2019-05-27 | 2024-02-27 | 东莞市汇成真空科技有限公司 | Coating machine for isolating target body through turnover target door |
Citations (3)
| 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 | Cell fusion device |
| CN86103459A (en) * | 1985-04-18 | 1987-11-18 | 澳洲生物科技公司 | Method for producing inhibin by using DNA recombination technology |
| CN1057482A (en) * | 1991-07-04 | 1992-01-01 | 北京理工大学 | The cell electricity merges, the electrotransfer device |
-
1993
- 1993-03-26 CN CN93103361A patent/CN1058291C/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN86103459A (en) * | 1985-04-18 | 1987-11-18 | 澳洲生物科技公司 | Method for producing inhibin by using DNA recombination technology |
| CN86108459A (en) * | 1985-12-11 | 1987-07-29 | 株式会社岛津制作所 | Cell fusion device |
| CN1057482A (en) * | 1991-07-04 | 1992-01-01 | 北京理工大学 | The cell electricity merges, the electrotransfer device |
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| Publication number | Publication date |
|---|---|
| CN1077495A (en) | 1993-10-20 |
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