CN1077495A - Cell modification technique with low energy ion beam and device - Google Patents
Cell modification technique with low energy ion beam and device Download PDFInfo
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- CN1077495A CN1077495A CN 93103361 CN93103361A CN1077495A CN 1077495 A CN1077495 A CN 1077495A CN 93103361 CN93103361 CN 93103361 CN 93103361 A CN93103361 A CN 93103361A CN 1077495 A CN1077495 A CN 1077495A
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- cell
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- ion beam
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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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Abstract
The present invention is cell modification technique with low energy ion beam and device.
The technology of the present invention is to carry out ise with the low energy ion beam ise to being placed on animal and plant cell in the microenvironment target chamber and the process precooling, and foreign gene imports, cytogamy etc.
Apparatus of the present invention are by an ion source, and a main vacuum chamber and the microenvironment target chamber that ionic fluid is passed through is formed.In the microenvironment target chamber specimen holder is housed, is furnished with the system of taking out in advance, the air-flow treatment facilities of high efficiency filter material and gas source interface is housed.Main vacuum chamber is connected by segregaion valve with the microenvironment target chamber.
Description
The present invention relates to biotechnology, international classification number C12N 13/00; G01N 1/00
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 at the mutagenesis of regional area to the damage of seed; Be energy exchange and quality deposition, 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, because ionic fluid and organism interact the spatial resolution of height is arranged on incident direction, if focusing of ion beam is below the micron bundle, just can realize the three-dimensional space resolution, 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 4～6 the degree, by sterilisable chamber, put into the microenvironment target chamber, the vacuum tightness of target chamber reaches 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
16～1 * 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
+Restraint insweptly, carry out cytogamy; After wall is by ion beam etching, carries out foreign gene and import
According to thermodynamic principles, the equilibrium temperature that vapour-liquid is practised physiognomy is relevant with vapour pressure, and when air pressure descended, the phase surface temperature can descend thereupon, supposes that a cell is that a radius is the little water droplet of γ, is exposed to 10 when its moment
-2During following vacuum, cell surface one deck will freeze rapidly, forming thickness is the nilas of dr, and As time goes on, nilas extends inward, the existence of nilas prevents the evaporation of internal moisture, and have enough intensity to bear negative pressure, and make the unlikely destruction of cell, be placed on overlong time in the vacuum chamber but work as cell, the nilas of cell surface layer will break for this reason because of bearing the negative pressure that does not live in the vacuum chamber, and 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 vacuum tightness 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
-6About, 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 (4)
1, a kind of cell modification technique with low energy ion beam, it is characterized in that be with the low energy ion beam ise to being placed in the microenvironment target chamber, and carry out sputter, etching through the animal and plant cell of precooling.
2, technology as claimed in claim 1 is characterized in that, the vacuum tightness in the microenvironment target chamber is 2 * 10
-4～2 * 10
-6Torr
3, technology as claimed in claim 1 is characterized in that the animal and plant cell before the ion sputtering etching, be with 0.5 the degree/minute speed be pre-chilled to the 4-6 degree
4, technology according to claim 1 is characterized in that:
When a. making cell mutation, inject N
+Ion;
When b. making the cell walls etching, use Ar
+Ion;
C. when two cells lean on very closely, use Ar
+Next inswept, carry out cytogamy, after cell walls is by ion etching, carries out foreign gene and import
D. a low energy ion beam cell decorating device is characterized in that, this device be by:
An ion source;
A main vacuum chamber, this chamber is furnished with the vacuum unit, and leaves the passage that can allow ion pass through, and a specimen holder is arranged in the vacuum chamber;
A microenvironment target chamber links to each other with big vacuum chamber by segregaion valve, and a specimen holder is arranged in the target chamber, also is furnished with one and takes out system in advance, an air-flow treatment facilities and the gas source interface that the high efficiency filter material is housed
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 true CN1077495A (en) | 1993-10-20 |
| CN1058291C 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 |
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|---|---|
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Cited By (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 |
| CN110295351A (en) * | 2019-05-27 | 2019-10-01 | 东莞市汇成真空科技有限公司 | A kind of coating machine that target body being isolated by convertible target door |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1020923C (en) * | 1985-04-18 | 1993-05-26 | 澳州生物科技公司 | Method for producing inhibin using dan recombinant technique using dan) |
| KR890003947B1 (en) * | 1985-12-11 | 1989-10-13 | 가부시기가이샤 시마즈세이사구쇼 | Cell fusion device |
| CN1025920C (en) * | 1991-07-04 | 1994-09-14 | 北京理工大学 | Cell electrofusion electrotransfer device |
-
1993
- 1993-03-26 CN CN93103361A patent/CN1058291C/en not_active Expired - Fee Related
Cited By (3)
| 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 |
| CN110295351A (en) * | 2019-05-27 | 2019-10-01 | 东莞市汇成真空科技有限公司 | A kind of coating machine that target body being isolated by convertible target door |
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1058291C (en) | 2000-11-08 |
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