CN1031649C - Bombardment device of gene with high-voltage discharge - Google Patents
Bombardment device of gene with high-voltage discharge Download PDFInfo
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- CN1031649C CN1031649C CN 90101042 CN90101042A CN1031649C CN 1031649 C CN1031649 C CN 1031649C CN 90101042 CN90101042 CN 90101042 CN 90101042 A CN90101042 A CN 90101042A CN 1031649 C CN1031649 C CN 1031649C
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 42
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- 238000013022 venting Methods 0.000 claims description 4
- 238000000034 method Methods 0.000 abstract description 19
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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 relates to a gene bombarding device discharging at high voltage, particularly to a device which brings external genes and accelerating micro-particles with a micro-bound method and bombards recipient cells in the engineering of biological heredity and variation. The present invention has wide uses in the recombination and conversion of cells. The bombarding device of the present invention is composed of a vibration absorbing base, a discharging chamber provided with a discharging electrode and a water tank, an emission spring inlaid with an O-shaped ring, a guard cap provided with an air exhasuting hole, movable target disc holder, a transparent outer cover with two segments, a safety cover, a high-voltage power source and a vacuum pump. The bombarding device can be placed positively, upside down, or sidewards. The present invention has the advantages of high bombarding efficiency, easily controlled energy, safety, reliability, convenient regulation and replacement of parts, and convenient experimental observation of parts.
Description
The invention belongs to variation and gene engineering, relating to a kind of is power with the electrion, quickens to contain the foreign gene micropartical, foreign gene is imported the device of acceptor (host) cell.
Biomutation in the present age and gene engineering are very active, and development is rapid, and one of them gordian technique is that the exogenic heredity gene is imported in the recipient cell, and method and apparatus for this purpose arises at the historic moment.The method of routine the earliest is the method for virus induction and chemical induction, and condition is that the recipient cell after birth allows the exogenic heredity gene to infiltrate, or recipient cell synthesizes with the cell that includes desirable exogenic heredity gene.Early stage this ordinary method has many shortcomings, and limits the use of this method in a lot of systems.
Method with perforation of electric field inducing cell and fusion has appearred in the eighties, and this is a kind of electrosynthesis, is with direct current (DC) pulsed field during beginning, is improved to later on and adopts the high power RF field, and the principle of the two is the same.In this method, DC pulse electric field efficient is low, finishing a transgenosis needs a large amount of acceptor (host) cells (reaching millions of times) usually, and there is the possibility that realizes the high-level efficiency transgenosis high power RF field, and might be applicable to the human gene therapy.But generally speaking perforation of electric field inducing cell and fusion method efficient are still not high, and can only carry out transgenosis to the cell that exsomatizes, and then it is implanted in the substance (being acceptor or host).
Recent years, the scholars of U.S. Cornell University have proposed the new ideas of " particle gun ", develop and quicken to contain the atomic bombardment device of gene with of foreign gene (being particle gun), have finished the experiment that foreign gene is imported biomass cells and tissue.The method and apparatus of the particle gun that the Cornell University proposes, compare with the chemical induction method with fusion method and virus induction more early with the electric field inducing cell perforation of above-mentioned routine, though purpose is identical, but be diverse new ideas, the method and apparatus that provides also is brand-new, particularly biomass cells and the tissue as acceptor (host) do not have the scope restriction, and the cell of any biology and tissue can.
The bombardment device of gene with that the Cornell University proposes has two types, people such as a kind of T.M.Klern of being invention with gunpowder (or pressurized gas) for propulsion source launch atomic particle gun (T.M.Klern etc., Neture, 1987,327:70-73); Another kind is arc-over rifle (PaulChriston etc., plant Physiol, 1988, the 87:671-674 that people such as PaulChriston and Dennis E.McCabe adopt; DennisE.McCabe etc., Bio/Technology, 1988,6:923-926.) yet, apply for patent of the present invention until us, above-mentioned two kinds of bombardment device of gene with also are experimental installation, there is not commodity selling, they have also just done the particle bombardment test to the plant in vitro tissue in the laboratory, issued brief message, more not have to disclose the structure and the experimental technique of the particle gun (bombardment device of gene with) of their use.With pressurized air is that the bombarder and the powder shotgun of power is basic identical, no longer carefully states herein.
Analyzing with gunpowder is that the particle gun in launch power source has following shortcoming, at first, the basic shortcoming that can't overcome fully is to pollute, discharge a large amount of flue gases and imperfect combustion particle after the gunpowder explosion, workspace is polluted, atomic clean level after this pollution may influence, and the workspace environment is degenerated; Next is that the control and the adjusting of emission rate is difficult to accurately, and can only be rough, fractionated; The 3rd, safety problem, gunpowder is inflammable and explosive hazardous substance, transportation, storage, use all are unsafe.
Another kind of bombardment device of gene with, it is the arc-over rifle of humans such as Paul Christon, its principle is that the shockwave that produces with high pressure arc discharge is a power, propelling is loaded with atomic film, use 100 purpose net barrier films before at acceptor (host or be referred to as target), and only make micropartical quicken to inject acceptor.It is reported that they test soybean with this arc-over rifle, condition be the discharge chamber diameter be 13 millimeters, with 25 kilovolts, the electrical condenser energize of 2 microfarads, operating voltage is 24 kilovolts and 14 kilovolts.Owing to do not see more detailed data, above-mentioned arc-over rifle be difficult to make accurately estimate.We imitate this condition and test, and found that the performance of this device is very unstable, are easy to generate to be launched the micropartical direction and to depart from, and influence the efficient of transgenosis, and key is the variation of film itself.The first, film is accelerated in the process, can not guarantee accelerating force uniform distribution on whole surface-area, that is to say, in fact is loaded with atomic film and is out of shape at the very start; Second, because the periphery and the discharge chamber of film must be fixed, so under the very large situation of acceleration, the center of film is inevitable moves earlier, and move the back all around, and film presents the deformation of central protuberance, again because the stressed of film periphery can not be the same, so that the deformation of film is very irregular, the contained micropartical of result breaks away from film in advance, and atomic transmit direction and speed all do not reach to be expected to set in advance; The 3rd, in most of the cases, because stop (particularly under the atmospheric condition) of gas have taken place to run into again before the deformation film in film, so be difficult to guarantee even propelling, the result is that certain part elder generation bump of film stops net, also causes the micropartical direction to depart from, and gene transfering efficiency greatly reduces.
In sum, early stage virus induction and chemical induction method are restricted because of its shortcoming use; Subsequently perforation of electric field inducing cell and fusion rule have consumption big, and efficient is low, and can only shift isolated cells; The particle gun and the method thereof of gunpowder emission metastatic gene have unsurmountable basic shortcoming at pollution, speed setting and secure context.At last, the arc-over rifle of new ideas has the workspace cleaning, energy size and speed setting controllability are good, the efficient height, safe and reliable advantage, but the structure of known arc-over rifle is not fully up to expectations, causes film unbalance stress generation deformation, cause the micropartical direction that is launched to depart from, influence gene transfering efficiency.
The objective of the invention is to overcome the shortcoming of prior art, a kind of new bombardment device of gene with high-voltage discharge is provided, it has the emission bullet of high efficiency discharge chamber and multiple pattern, the door that the deft design distance is adjustable, guarantee safety screen safe in utilization, and simple and practical target dish frame etc., whole device is safe, reliable, efficient, can just put, inversion or side work, realize high efficiency transgenosis.
Bombardment device of gene with high-voltage discharge of the present invention (hereinafter to be referred as bombardment device of gene with or bombarder) is by base, and discharge chamber is launched bullet, door, and safety screen, target ware, target dish frame, outer cover, cushion blocking and high-voltage pulse power source and vacuum pump are formed.
The discharge chamber of bombardment device of gene with of the present invention is contained on the base, discharge chamber is cup-shaped, discharge electrode is contained in the bottom of discharge chamber cavity, adhere to atomic emission bullet and be contained in the open section of discharge chamber, the emission bullet before (on) side restricted emission bullet stroke door, door preceding (on) side is useful on the target ware of adhesion receptor (host), acceptor is as the atomic target that hits, the target ware is contained on the transportable target dish frame, and above working portion is housed in the outer cover, by high-voltage pulse power source discharge chamber is powered, there is vacuum tunnel to be communicated with vacuum pump in the outer cover on the base, be pumped into true chamber during work in the outer cover, also can not vacuumize, under the atmospheric environment of cleaning, work.
Fig. 1, bombardment device of gene with high-voltage discharge of the present invention
Fig. 2, several emission bullet structures
Fig. 3, three kinds of door structures
Fig. 4, target dish frame
Describe details of the present invention and embodiment in detail below in conjunction with accompanying drawing.
In the embodiment of the invention, main parts size is contained on the base 1 as shown in Figure 1.Base is a plate-like, makes with insulating material, and upper surface has the sealing groove that matches with outer cover 14 lower surfaces, dress sealing-ring 17 in the groove.There is cavity lead-in wire chamber 23 bottom surface.Dress conducting rod 2 in the shoulder hole of vertical direction, cover round section joint ring 3 on the boss in conducting rod stage casing is with the shoulder hole formation sealed structure on the base.Concentric cable 19 puts in the lead-in wire chamber 23 of base bottom, links with the conducting rod lower end.The cable outlet end does not contact base, makes shoulder hole play the effect that improves surface insulation intensity.Cushion blocking 21 is equipped with in the base plate 20 chamber capping that will go between under the base plate.Vacuum tunnel 22 is arranged on the base, be connected with vacuum pump.High-voltage pulse power source and vacuum pump do not draw in the accompanying drawings.
Discharge chamber 5 is goblets that there is one section double-screw bolt the lower end, is contained in this section double-screw bolt in the screw of base 1.The discharge chamber inner chamber is up big and down small shoulder hole, and discharge electrode 6 parallel opposed are contained in the bottom of inner chamber.Between these intracavity bottom two discharge electrodes, the water-bearing channel that forms conducting bridge is arranged, (need in this water-bearing channel, splash into the water of trace during work), with guarantee bombarder just put, under inversion or the side state, the minor amount of water that splashes into can both exist in the groove, form between two discharge electrodes conducting bridge and can works better.Discharge electrode is made screw shaped, uses nut locking after discharging gap mixes up.Discharge electrode leading-out end and conducting rod 2 upper ends connect with on line 7 and tighten up with nut.The epimere of shoulder hole).The internal diameter of open section is made different sizes and is matched with the emission bullet.Discharge chamber is made with insulating material.
Emission bullet 8 is right cylinder or " T " font body (latter's longitudinal section is " T " font).Fig. 2 is several typical emission bullet structures.The outer diameter D of emission bullet matches with discharge chamber open section internal diameter, and no matter bombarder is just put, is inverted or side, and the emission bullet can both normally be placed in the open section of discharge chamber.Emission bullet 8 usefulness insulating material are made, and can use the engineering plastics injection-compression molding when quantity is big.
In order to improve emission efficiency, emission plays the cylindrical surface hypomere and is embedded with " zero " shape circle 9 (Fig. 2-2 and Fig. 2-3), when the emission bullet is the T font such as Fig. 2-4.Zero shape circle 9 makes and forms sealing between emission bullet and discharge chamber open section, but sealing is too not tight, and the high pressure draft that spills along the slit that the emission bullet cooperates with the discharge chamber open section was reduced, and also can not increase the resistance that the emission bullet quickens.Thereby the top speed of emission bullet is improved.T type emission bullet also is the high pressure draft that is spilt with extended T shape edge joint, has both utilized this air-flow to make the emission bullet improve acceleration, also makes this air-flow change impact direction.It is smooth bright and clean that emission plays end face, above armed micropartical sticks to.When micropartical did not need too high speed, emission plays end face also can setting-in zero shape circle 10 (Fig. 2-3), and to the bump of door 11 and reduce atomic speed, this also is an approach of governing speed with buffering emission bullet.
Safety screen 4 is housed on the base, discharge electrode leading-out end, conducting rod upper end and on line 7 are covered on wherein, only discharge chamber top is exposed in the upper end open of safety screen together with the emission bullet.Safety screen is made with insulating material.
Fig. 3 is several structures of door 11.Door 11 is contained on the top of safety screen 4, is used for stopping emission bullet 8, the stroke of restriction emission bullet, and only allow acceptor (host) cell tissue on the micropartical directive target ware 12 that contains foreign gene.Door 11 is that top is that center foraminous disk and bottom are the molectron of cylinder substantially, and the internal diameter of its underpart (cylinder) opening is bigger than the external diameter of discharge chamber, and discharge chamber mouth and emission bullet are all covered on the inside; The diameter of top (disk) centre hole is launched bullet like this and is struck door and stopped less than the diameter of emission bullet, breaks away from the emission bullet and stick to the micropartical that emission plays end face, by centre hole directive acceptor (host) cell.Radially have a circle (Fig. 3-2) or two circle (Fig. 3-3) venting holes 29 (also can be split into seam) on the door cylindrical wall, its effect is when avoiding electrion, and the blast of rushing out from discharge chamber is directly to the impact of target ware 12.Upper end one circle venting hole is opened the bottom at cylinder Nei Ding top and outer disc, rise simultaneously and allow the air that pushed by the transmitted at high speed bullet before the emission bullet enclose venting hole to discharge rapidly from this, do not form the air cushion between emission bullet and the door, influence atomic launching effect, particularly important when this launches under normal atmosphere (not vacuumizing) environment.Door 11 end openings have screw thread, can regulate the height of door end face range transmission bullet end face according to the precession degree of depth, make every effort to launch bullet and clash into the door end face when top speed, to obtain minimum best again emission bullet stroke.
Target dish frame 13 as shown in Figure 4 is target ware 12 to be installed and to be regulated target ware position.The target ware is a plate-like, is used for adhesion receptor (host) cell tissue.Target dish frame is made with thin lath, light and handy being suitable for.Article three, resilient lath 25 is connected with Circular Plate 24 with the stage casing, and lath can be unequal to the two sections size of Circular Plate.Rubber pad 26 is equipped with at two of lath, and the circumscribed circle diameter of two groups of rubber pads is slightly larger than the internal diameter of outer cover 14 up and down.Plate-like target ware 12 can be contained in the hypomere (as shown in Figure 1) of target dish frame 13 and hold with rubber pad, also can be placed on the Circular Plate 24.Target dish frame utilizes the frictional force between elasticity and rubber pad and outer cover to be contained in the outer cover, can move up and down adjusting, stops on the arbitrary height in the outer cover.
Outer cover 14 is by two sections transparent tube 28,27, and top cover 15 and vacuum rubber sealing-ring 16,18 are formed.Sealing-ring 16 is contained between top cover 15 and the last transparent tube 28.Sealing-ring 18 is contained between the two sections transparent tube 28,27.The lower surface of outer cover is pressed on the rubber gasket 17, discharge chamber, emission bullet, safety screen, door, target ware and the whole working portions of target dish frame are covered on wherein, be evacuated during work, both avoided pollution and the interference of outside atmosphere, improved emission efficiency again workspace.In the environment of cleaning, not vacuumizing also can works better.Transparent tube and top cover adopt transparent synthetic glass or glass to make, and operational observations is convenient.
Bombarder of the present invention can just be put (as shown in Figure 1), inversion or side work.Base is descending at last, top cover during inversion, and cushion blocking also changes the installation site thereupon.If be necessary, bombarder can side work, as be used for biological (or human body) substance original position cell is bombarded, and realizes the importing of foreign gene.At this moment without the last transparent tube 28 in the outer cover 14, and will descend transparent tube 27 to make the form that matches with the substance original position, and directly cover on the former bit position of substance and get final product.
The operation and the working process of bombarder of the present invention are fairly simple, above-mentioned explanation chat to some extent and, the existing operating process that it is complete is summarized as follows.At first each parts with bombarder carry out aseptically process, assemble and place in the aseptic cage (chamber) stand-by then.Take off outer cover 14 (comprising transparent tube 28 and 27 up and down) during the work beginning, unload door 11, (promptly between two discharge electrodes 6) splash into micro-purified water (about 1 milligram) in the conducting bridge water-bearing channel in discharge chamber 5; With select for use, end face adhered to the micropartical emission bullet 8 that contains foreign gene and inserted in the discharge chamber open sections, screw on door 11 and be adjusted to selected in advance position after tighten; Load onto down transparent tube 27, the target ware 12 that will be placed with acceptor (host) cell is contained on the target ware support 13, then target ware support 13 is placed to the predetermined position in the transparent tube 28, will go up transparent tube 28 again and cover on down on the transparent tube 27; The errorless back startup of inspection installation vacuum pump vacuumizes and reaches 0.1 normal atmosphere; Seeing through transparent housing checks again, if all are normal, start high-voltage pulse power source, charging boosts to previously selected magnitude of voltage (need approximately 10-20 second), at this moment the high pressure of high-voltage pulse power source is not communicated with discharge electrode 6 in the bombarder discharge chamber, observation and inspection once more, all are normal to confirm bombarder; Press high-voltage pulse power source igniting (bombardment) button, high pressure promptly is added on the discharge electrode 6 in the bombarder discharge chamber, produce high pressure pulse discharge, emission bullet 8 penetrates the top that impinges upon door 11 then, the micropartical that emission plays end face carries foreign gene by door centre hole directive acceptor (host) cell or tissue that scatters, and foreign gene imported in acceptor (host) cell finishes bombardment; Open the vacuum valve that is connected with vacuum pump, eliminate vacuum, can take out target ware and acceptor (host) tissue after taking off transparent tube 28.So far, the operation that once imports foreign gene of using this bombarder to carry out is all finished.Wherein, the time that the high-voltage pulse power source charging is boosted is 10-20 second, and the time that is added on the bombarder is to be several microseconds the discharge pulse time, and the work of bombarder is very safe, reliable, efficient.
Bombarder of the present invention has following outstanding feature and outstanding effect:
(1) provide brand-new, high efficiency bombardment device of gene with high-voltage discharge, bombarding energy is easy to control, can stepless change regulates, and the workspace cleaning is pollution-free, and is safe and reliable, simple and compact for structure, and overall performance and efficient greatly improve.
(2) provide the emission bullet structure of high efficiency discharge chamber and multiple pattern, formed the water-bearing channel of conducting bridge between discharge chamber bottom and the discharge electrode, work is practical reliable; Emission plays setting-in zero shape circle and tightly cooperates with discharge chamber, the emission efficiency height, and it is extremely short that emission plays stroke; Transmit direction does not depart from, and gene transfering efficiency greatly improves.
(3) door deft design, impact wave is to the direct impact of target ware when having eliminated discharge, particularly door and some emission bullet are (as the T font, or end face has zero shape to enclose) be used in combination, not only the blast (air-flow) during electrion changes impact direction, direct impact target ware makes the emission rate of emission bullet be improved again and controls.
(4) target dish frame structure uniqueness is simple and practical, and is easy to adjust.
(5) there is the high-voltage part discharge chamber, discharge electrode and lead-in wire chamber etc. in short-term by sealing cover, guarantee safe and reliable.
(6) transparent housing is divided into two sections, and parts such as door, emission bullet, target dish frame regulate and change conveniently, is convenient to operation and observes.
(7) as required, bombarder can just be put, inversion or side use, and can vacuumize work, also can under atmospheric pressure work, and is applied widely, particularly can realize the foreign gene importing to substance biology in situ tissue.
Adopt bombardment device of gene with high-voltage discharge of the present invention in several experiments such as yeast cell conversion process, all to succeed.
More than chat and one embodiment of the present of invention in, be 6 centimetres target ware and general requirement of experiment for adapting to diameter, the base diameter of bombarder is 12 centimetres, 32 centimetres of bombarder height overalls, about 1 centimetre of discharge chamber bottom internal diameter, emission plays about 2 centimetres of diameter D.The LDZ1/10 type high-voltage pulse power source that supporting with it discharge power supply adopts CAS Electrical Engineering Research Institute to produce, operating voltage 5-10 kilovolt, energy region is the 100-1000 joule.
Claims (6)
1, a kind of bombardment device of gene with high-voltage discharge, by high-voltage pulse power source discharge chamber is powered, working portion covers in the outer cover, comprising target ware and target dish frame, vacuum tunnel will be communicated with vacuum pump in the outer cover, it is characterized in that the cup-shaped discharge chamber is contained on the base, and discharge electrode is contained in the bottom of discharge chamber cavity, adhere to atomic emission bullet and be contained in the open section of discharge chamber, emission plays the door of the place ahead restricted emission bullet stroke.
2, bombardment device of gene with as claimed in claim 1 is characterized in that the discharge chamber inner chamber is up big and down small shoulder hole, has water-bearing channel between the discharge electrode of intracavity bottom parallel opposed.
3, as claim 1 and 2 described bombardment device of gene with, it is characterized in that launching bullet is right cylinder or T font body, and its outer diameter D matches with discharge chamber open section internal diameter, and emission plays the cylindrical surface hypomere and is embedded with round section joint ring, and end face also can the setting-in round section joint ring.
4, as claim 1 or 3 described bombardment device of gene with, it is characterized in that door is the molectron of center foraminous disk and cylinder, the diameter in hole, its top (disk) radially has a circle or two circle venting hole or seams less than the diameter of emission bullet on the cylindrical wall of bottom.
5, as claim 3 or 4 described bombardment device of gene with, it is characterized in that the target dish frame structure is that three resilient laths are connected with Circular Plate with the stage casing, lath can be unequal to the two sections size of Circular Plate, and rubber pad is equipped with at two of lath.
6,, it is characterized in that the safety screen on the base covers on discharge electrode leading-out end, conducting rod upper end and on line wherein as claim 1 or 3 or 5 described bombardment device of gene with.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 90101042 CN1031649C (en) | 1990-03-09 | 1990-03-09 | Bombardment device of gene with high-voltage discharge |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 90101042 CN1031649C (en) | 1990-03-09 | 1990-03-09 | Bombardment device of gene with high-voltage discharge |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1054796A CN1054796A (en) | 1991-09-25 |
| CN1031649C true CN1031649C (en) | 1996-04-24 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 90101042 Expired - Fee Related CN1031649C (en) | 1990-03-09 | 1990-03-09 | Bombardment device of gene with high-voltage discharge |
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| Country | Link |
|---|---|
| CN (1) | CN1031649C (en) |
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1990
- 1990-03-09 CN CN 90101042 patent/CN1031649C/en not_active Expired - Fee Related
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| Publication number | Publication date |
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| CN1054796A (en) | 1991-09-25 |
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