Title of Invention

PARTICLE ACCELARATION APPARATUS

Abstract The Gene pro 2000He is a laboratory apparatus. The subject matter of the invention is an apparatus for genetic transformation in Biotechnology. The transformation of genetic material is achieved through the mechanism of particle acceleration. It involves high velocity acceleration using pressurized helium in conditions created by vacuum under certain controlled operating parameters. The acceleration enables penetration of DNA coated micro projectiles (Tungsten particles) into the cell wall and membrane. Here the acceleration is fully controlled to absolute and safe levels. The apparatus guarantees safety. It is also reliable. There is negligible or no damage to the cells. The success rate is very high. Gene pro 2000He reduces operational time for the transfer of foreign DNA into plant cells. It is a low cost apparatus and easy to operate with three distinct units, which simultaneously work with all indications about the progress of the operations automatically, displayed. This invention is a class by itself in its operational parameters most conveniently designed after great and dedicated research work and experiments. The inventive steps are clearly described in the final specification and so are the claims.
Full Text

Complete Specification
GENE PRO 2000He Apparatus - Particle Acceleration
Apparatus for genetic transformation
1. PREAMBLE:
This invention relates to accelerated, safe and economical mechanism for transfer of foreign DNA into plant cells.
2. Where DNA transfer system operates on biolistic principles, it involves high velocity acceleration enabling penetration of DNA coated micro-projectiles (tungsten particles) into the cell wall and membrane using pressurised helium in conditions created by vacuum and under certain controlled operating perameters.
3. In GENE PRO 2000He, the Particle accelerating mechanism is achieved by reducing the operational time for the transfer of foreign DNA into plant cells through a special and compact apparatus scientifically designed as an independent instrument and distinct by itself.
4. GENE PRO 2000He, makes the acceleration fully controlled to absolute safe levels.
5. GENE PRO 2000He, is economical as it uses locally available low cost materials for its construction.
6. GENE PRO 2000He, is reliable. Safety is assured by an electronic control station which contains suitable controls for the release of electricity, gas and vacuum. The station contains all appropriate operational meters and displays electronic indications.
7. KNOWN ART:

(a) As is known in the art, genetic variation in plants which is necessary for crop improvement for getting higher yields, for securing immunity against natural hazards, for building resistance to, pests and diseases and for avoidance of weeds, is generated through hybridization, mutagenesis and polyploidy. More recently, biotechnological tools have become available for producing transfer of specifically constructed gene construct through various transformation techniques which broadly constitute Genetic Engineering.
(b) The plants obtained from genetic engineering embody in them gene or genes usually from unrelated organisms. Such genes are called Transgenes and the plants containing transgenes are known as transgenic plants. Transgene plants have both basic and applied uses. They are extremely useful in studies on plant molecular biology, regulation of gene action, identification of regulatory sequencies etc. Genetic Engineering is also considered as an efficient approach for improving and creating agronomically valuable traits.
(c) The uptake of foreign DNA by plant cells is called Transformation. A variety of techniques are used to introduce transgenes into plant cells and these can be grouped into the following two categories :


(d) In the later category various methods of direct gene transfer are available as listed below : (1) Chemical methods, (2) Electroporation, (3) Particle gun delivery, (4) Micro injection, (5) Pollen transformation, (6) laser induced, (7) Fibre-mediated gene transfers etc. Particle gun delivery also called Biolistic or Ballistic method of DNA delivery is used for transformation. It has general applicability to plant species and can be used for delivering DNA into virtually all tissues. An advantage of particle bombardment is that the physical penetration of DNA into the plant cell entails gene transfer independent of regeneration ability of the species. It can be used to transform shoot apical meristems, leaf blades, immature and mature embryos, mature pollen, root and shoot sections etc..
In this method, 1 -2 ji.m. tungsten or gold particles coated with the DNA to be used for transformation are accelerated to suitable velocities which enable their entry into plant cells/ nuclei. Particle acceleration is achieved by using a device which varies considerably in design and function. Biolistic technique has been used to produce stable gene transfers in Cotton, Maize, Rice, Sugarcane, Tobacco, Wheat, Papaya, Poplar, etc. and is an attractive method applicable to all plant species and even to animal cells. But the main limitation is the requirement of a costly specialised acceleration device which happens to be complex also. The need to develop a low cost and efficient ' device that is user friendly, clean, safe, simple to operate and less time consuming would aid in the much needed utilization of the technology. Innovations in this direction would be of great support to researchers and students in Universities. The present research effort, and the inventive steps aim at minimal damage to the plant cells. The irmovations introduced in GENE PRO 2000He, describes and listed below achieve all the above objectives.
8. This invention is described with reference to the accompanying figures 1,2 and 3 a schematic representation of the particle acceleration apparatus "GENE PRO 2000He".
Detailed description of the parts and their function.
The present invention GENE PRO 2000He has in all 36 parts. Parts 1 to 8 are connected with what is called Vacuum Chamber. Parts 29 to 36 are the parts in what is called Particle bombardment device. Parts 9 to 24 & 26 are located in what is called control station. Part 25 is Vacuum Pump. Parts 27,28 are helium gas cylinder and its regulator. Finally parts 37 to 42 are illustrations as to what happens in Vacuum Chamber after helium gas is released.
Particle Bombardment device a sectional view of which is presented in Fig 2 is assembled by vertical axis on the top of Vacuum Chamber (1) located in the centre of the collar (34). Particle Bombardment device has a main function of generating a powerful blasting effect to propel pressurized helium gas to transport DNA coated micro projectiles to introduce them into target cells. The gas portal (29) is situated at the entry of gas, close to solenoid has to accomplish mainly two important functions, to contain fluctuations while receiving pre set high pressure helium gas from the cylinder which is required to generate powerful bombardment and to maintain constant built in line pressure and to absorb sudden instant returning shock waves of gas arising during bombardment and due to exhausted gas.
Fluctuations in line pressure have been observed in the absence of the gas portal due to gas consumed for bombardment which are eliminated in their apparatus by introducing gas portal (29) to augment the efficiency of the apparatus.
A needle valve (30) is fitted in between the gas portal (29) and the solenoid valve(32) to manoeuvre the flow of gas into solenoid according to user requirements.

A special 2 way NC solenoid high pressure valve (32) which is capable of withholding the force of high pressure helium gas and sensitive to open rapidly when activated electro magnetically by timer relay (14) to generate the best powerful bombardment instantaneously, is installed.
The coupling (33) contains a passage for gas flow in the centre, and another passage connected to a nipple for evacuating the vacuum chamber (1) Particle bombardment device is assembled in the location aligned carefully with vertical axis and concealed (31).
At the internal top of the vacuum chamber (1) just below the coupling (33) which is at the end of the gas passage, the leur- lok adaptor (8) is connected where filter holder could be readily attached to and removed.
Helium gas cylinder (27) under high pressure is employed as a source or kinetic energy to provide necessary gas supply required for the system. The chemical properties of helium gas is well under stood which is light in weight and inert by nature and having a characteristic of high speed expansion. Helium gas with high purity i.e. 4.6(99.996%) could be used for application.
A special high pressure two stage regulator (28) which is suitable for the application having appropriate gauges for displaying cylinder pressure and applied pressure, is fitted to the cylinder and connected to the vacuum chamber (1) with suitable hose piping through the control station for. generating necessary bombardment.
A portable Vacuum pump (25) is connected to Vacuum chamber (1) via control station (9) with suitable hose piping to evacuate the chamber before bombardment and can be released after bombardment.
The control station (9) monitors complete operations of the apparatus consists of vacuum control valves (10,11) and absolute gauge (18) for vacuum, absolute gauge (17), for gas. Timer relay (14), push button switch (15) and alarm(l 6) to activate the solenoid(32) are found in the control station. Power ON/OFF (13), key switch (12), for security. Glow switch for vacuum pump ON/OFF (21) power ON/OFF display (20), ready display(19), for indicating the status of the system are provided on the front panel. Necessary power sockets gas/vacuum connecting nipples and safety fiise are located on the rear panel of the control station.
The control station with integrated power circuits and controls would complement the users' convenience and comfort. It also gives him control over the whole system without any mobility. Thus it helps to enhance productivity and efficiency of the system. The security key switch ensures the user safety and security of the apparatus in that the user only would be able to use his apparatus without any unauthorised interference by any body else.
9. DESCRIPTION OF THE DRAWINGS
The graphic representation of the present invention will be more clear if it is seen with reference to the attached drawings Fig-1, Fig-2 and Fig-3.
Fig-1 is a schematic view of the complete set up of GENE PRO 2000He particle acceleration apparatus for particle bombardment and all its necessary accessories like helium gas cylinder with regulator, vacuum pump, control station.
Fig-2 is a Sectional view depicting cross section of the functional parts of the particle bombardment device showing the passages of gas and vacuum and the details of the coupling.
Fig-3 is a typical view of the reaction that occurs as a result of bombardment depicting the wide spread pattern of the DNA coated microprojectiles released from the tip of the filter holder, propelled with high velocity towards the target cells/tissue and introduced into the cell membrane.

10. OPERATION :
Particle suspension is placed on the screen within the filter holder (5) by opening and re assembled. It is screwed to the leur-lok adaptor. The petri dish containing plant tissue is placed on adjustable tray (4) in vacuum chamber at suitable distance from the filter holder. The tissue is covered with baffle (6) to prevent spillage of particle suspension and dispersion of tissue. Closing the door, a vacuum of 600 mm/Hg is applied and particles are discharged through filter holder and through baffle direct into tissue when helium is released following activation of solenoide (32) by the timer relay (14). The operation over, the vacuum is released to open the door of vacuum chamber and the tissue thus duly bombarded with particles is removed.
11. INVENTIVE STEPS :
(a) The process involves the high velocity acceleration of DNA coated micro-projectiles (tungsten/ gold particles) using pressurised helium in combination with a vacuum which enables their penetration directly into the cell wall and membrane of the plant cells, through a special mechanism created in the apparatus to reduce tissue damages.
(b) The second part of the invention is the special mechanism created in the apparatus comprising gas portal, needle valve, solenoid, filter holder, and baffle.
(c) The third part of the invention dispenses totally with what are called macro-carriers and instead provides a direct and safe delivery mechanism through filter holder.
(d) The fourth part of the invention relates to the identification of suitable indigenous metals, alloys components and spares used in the construction of this instrument to make it cost effective without effecting quality of operation.
(e) The fifth part of the invention relates to making the instrument quickly available for repeated bombardments so that its turnover and productivity are nearly doubled.
(f) The sixth part of the invention relates to hygenic aspect of the operations by which clewing operation need not be undertaken for each delivery.
12. HIGHLIGHTS-ADVANTAGES OF THE GENE PRO 2000He :
i) Efficient and compatible with biological targets.
ii) A vacuum chamber was used to reduce the drag on the particles.
iii) A timer-relay-driven solenoid provided more consistent acceleration by permitting better control of the amount of helium released through the use of a timer-relay.
iv) A gas portal was attached to the entry side of the solenoid. The volume of helium released to accelerate the particles could be further controlled by gas portal at the upstream of the solenoid thereby reducing tissue damage/displacement due to sudden impact of bombardment.
v) The flowing helium accelerates particles directly in a stream of the low pressure helium.
vi) Cost effectiveness : The overall cost of the instrument is l/5th of the price of international models contemporary and affordable by Indian users.
vii) The absence of macrocarriers reduced consumables, cleanup time and cycle time, thus increased productivity. In addition to this the device gave transient transformation of a variety of different plant tissues.





13 As an instrument to be used in the laboratories, its usefulness is beyond all doubts, There are immense market opportunities.
14. We claim :
Claim 1 : The entire apparatus in all its essential parts like the gas portal, solenoid valve and filter holder integrated into it, its structure which is scientifically determined called GENE PRO 2000He.
Claim 2 : The new and novel automatic device embedded into the said apparatus by which foreign DNA is lead into the recipient cell with safety, certainty and with little or least damage to the cell.
Claim 3 : The new and novel automatic device by which the implantation of DNA into the recipient cell is achieved by direct drive into cell and cell membrane, regulated from gas portal through filter holder.
Claim 4 : The new novel device by which the vacuum chamber is so arranged that the impediment necessary to the flow of helium gas mixed with DNA coated tungsten particles to reach the target without or minimum dispersion.
Claim 5 : The conception and arrangement of source equipment for power, gas and vacuum to sustain the principal part of the apparatus which all work in synchronisation.
Claim 6 : With reference to claim 1, the gas portal, solenoid valve and filter holder their placement and their special function.
Claim 7 : With reference to claim 2 the baffle placed over the tissue, its placement and special function.
Claim 8 : With reference to claim 1,2,3, and 5 the procedure and process by which when the apparatus once set, it works automatically and with full synchronisation of all its parts.


LIST OF PARTS
FIG-1 FIG 2
1. Vacuum Chamber 29. Gas Portal
2. Door 30. Needle Valve
3. Target shelf 31. Protective Housing
4. Target tray 32. Solenoid
5. Petridish with target tissue 33. Coupling
6. Baffle 34. Collar
7. Filter holder 35. Top of vacuum chamber
8. Leur-Lok adaptor 36. Vacuum connection
9. Control station FIG-3
10. Needle Valve - Vacuum charge 37. Microprojectiles
11. Needle Valve - Vacuum release 38. Filter screen
12. Key switch - Security 39. Tip of filter holder
13. Power ON/OFF Switch 40. Gas stream
14. Timer relay 41. Microprojectiles propelled towards target
15. Shoot switch (Push button switch) 42 Target cells/Tissue.
16. Alarm
17. Absolute gauge- Gas
18. Absolute gauge - Vacuum
19. Ready - display
20. Power ON/OFF Display
21. ON/OFF switch - Vacuum pump
22. Hose for vacuum
23. Power cord
24. Hose for gas
25. Vacuum pump
26. Power plug
27. Helium gas cylinder
28. High pressure special regulator

Documents:

391-mas-2000-abstract.pdf

391-mas-2000-claims filed.pdf

391-mas-2000-claims grand.pdf

391-mas-2000-correspondnece-others.pdf

391-mas-2000-correspondnece-po.pdf

391-mas-2000-description(complete) filed.pdf

391-mas-2000-description(complete) grand.pdf

391-mas-2000-description(provisional).pdf

391-mas-2000-drawings.pdf

391-mas-2000-form 1.pdf

391-mas-2000-form 19.pdf

391-mas-2000-form 26.pdf

391-mas-2000-form 4.pdf

391-mas-2000-form 5.pdf


Patent Number 204512
Indian Patent Application Number 391/MAS/2000
PG Journal Number 26/2007
Publication Date 29-Jun-2007
Grant Date 22-Feb-2007
Date of Filing 23-May-2000
Name of Patentee SHRI. K. DURGA VARA PRASAD
Applicant Address S/O K. SATYANARAYANA, NO. 205, PRASHANT APARTMENT , SUBHASSH NAGAR SRINAGAR COLONY , HYDERABAD 500 073
Inventors:
# Inventor's Name Inventor's Address
1 K.DURGA VARA PRASAD S/O K. SATYANARAYANA, NO. 205, PRASHANT APARTMENT , SUBHASSH NAGAR SRINAGAR COLONY , HYDERABAD 500 073
PCT International Classification Number C 02 M 15/63
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA