WO1993007256A1

WO1993007256A1 - Particle gun for introducing dna into intact cells

Info

Publication number: WO1993007256A1
Application number: PCT/US1992/008466
Authority: WO
Inventors: James Richard Wong; Paul Bellamy
Original assignee: Ciba-Geigy Ag
Priority date: 1991-10-07
Filing date: 1992-10-05
Publication date: 1993-04-15
Also published as: AU2781892A

Abstract

A particle gun for introduction of genetic material into cells, tissues or organs uses gas propulsion from a compressed gas reservoir to propel the particles. Other features of preferred embodiments include a stripper for stopping the particle carrier outside the bore of the gun to allow easy reloading. A method of using the gun is also disclosed.

Description

Particle Gun for Introducing DNA into Intact Cells

Technical Field

This invention is directed to genetic manipulation of cells and is particularly directed to microinjection of DNA or other genetic material into intact cells.

Background

The process of particle bombardment for genetic transformation of plant cells was originally demonstrated with an apparatus powered by a gun powder charge. This apparatus, which is now commercially available for a variety of transformation uses, accelerated the particles on a carrier block that was stopped abruptly, allowing the inertia of the small carrier particles to carry them forward into the target cells. However, use of gunpowder to produce the propulsion force in the gun causes several problems. The amount of force cannot be varied. Gunpowder usage is inherently dangerous. A large column of hot gases and a large amount of soot particles from the combustion are produced, some of which can be carried to the target chamber. The apparatus must be taken apart and cleaned between shots and the disposable parts replaced, which can take several minutes. The disposable parts are also relatively expensive. In addition, a large target area is bombarded, usually several centimeters in diameter, which is a disadvantage if the target tissue is small.

A second guns design uses a different motive force, namely gas generated by electrical discharge through a droplet of water, a different geometry, and a different method of particle preparation. The second gun works at a fixed, very short distance and uses a high voltage electric arc discharge which is hazardous.

"A

Compressed gas-driven guns have been described. A third gun uses a timing circuit to meter out a portion of propulsion gas but does not give a reproducible shot A fourth gun is commercially availabe as a supplement to the gunpowder gun. In this device, a chamber sealed at one end by a plastic rupture disk is slowly pressurized with compressed gas until the plastic deforms to the point of breakage, whereupon a wave of gas is suddenly released. This wave strikes another plastic sheet which bears the microprojectiles, propelling them towards the target tissue. This design also has reproducibility and reliability problems, since individual rupture disks do not break at constant or predictable pressures, and the point of rupture varies over the surface of the disk so that the gas wave is not symmetrical nor coaxial with the target Sometimes the disk balloons out rather than rupturing even at full pressure resulting in a dangerous, pressurized condition known as a "hang-fire." Disks are available in only a few discrete thicknesses which limits the range and intervals between usable gas pressures.

It is therefore an object of the invention to provide a gun which has a rapid firing cycle and a consistent force and accuracy of the shots fired. Another object of the invention is to provide a simpler gun which uses a controlled, reproducible, adjustable and safe propulsion source. Yet another object is to provide a gun which is particularly suitable for small targets, such as tissue only a few millimeters in diameter (or even smaller).

Relevant Literature

Particle guns are described in U.S. patent 4,945,050, European patent 270, 356, and Morikawa et al. (1989) Appl. Microbiol. Biotechnol. 31: 320-322.

Summary of the Invention

The present invention is directed to a particle gun for the introduction of particles carrying genetic material into intact cells, comprising:

an elongated chamber having a principal axis with an opening located at one end of said principal axis, said chamber having an empty bore portion of invariant cross section centered on said principal axis; a stripper means located on said principal axis between said target holder and said bore opening, said stripper means having an opening therethrough of a cross section smaller than that of said bore portion; a deformable flying block adapted to move along said principal axis in said bore portion while substantially blocking passage of compressed gas past said flying block in said bore portion of said chamber, said flying block being too large to enter said stripper means opening, wherein a surface of said flying block proximal to said opening when said flying block is in said bore is adapted to hold said carrier particles and wherein said flying block is moveable by a compressed gas and yet stoppable on contact of the flying block with said stripper means; means for introducing said flying block into said chamber; an inlet for compressed gas located in said chamber behind said flying block and distally in said bore portion relative to said opening; means for controlling access of a compressed gas to said inlet; and means for maintaining said compressed gas at a constant volume and pressure prior to release into said chamber by said means for controlling access; whereby when said carrier particles are loaded onto said flying block and cells are present on a target in said target holder, entry of compressed gas into said chamber causes said flying block to travel along said axis until said flying block is stopped by said stripper means, thereby releasing said carrier particles which continue to travel through said stripper means opening into said cells.

The invention also comprises a method for the introduction of genetic material into intact cells, wherein said genetic material, which one desires to have introduced into said intact cells, is prepared, wherein ballistic means are used for accelerating said genetic material at a velocity sufficient to enter into said intact cells and thereby transform them, without damaging said cells, and wherein shoots and whole plants are generated from said transformed cells.

In a preferred embodiment the method for the introduction of carrier particles carrying genetic material into intact cells comprises: loading said carrier particles onto a deformable flying block in an elongated chamber having a principal axis and an opening located at one end of said principal axis, said chamber having an empty bore portion of invariant cross section centered on said principal axis, wherein said flying block is adapted to move along said principal axis in said bore portion while substantially blocking passage of compressed gas past said flying block in said bore portion of said chamber, a stripper means located on said principal axis between said target holder and said bore opening, said stripper means having an opening therethrough of a cross section smaller than said bore portion; said flying block being too large to enter said stripper means opening and a surface of said flying block proximal to said bore opening is adaped to hold said carrier particles and wherein said flying block is moveable by a compressed gas and yet stoppable on contact of said flying block with said stripper means thereby releasing said particles to travel through said stripper means opening into said cells; loading said cells onto a cell carrier target holder located outside said chamber on said principal axis and adjacent said opening; and injection a constant volume of compressed gas into said chamber behind said flying block and distally on said bore relative to said opening; whereby entry of compressed gas into said chamber causes said flying block to travel along said axis until said flying block is stopped by said stripper means thereby releasing said carrier particles continue to travel through said stripper means opening into said cells.

A constant volume of compressed gas can be obtained, for example, by charging a chamber with compressed gas from a source and maintaining said gas at a constant volume and pressure; and charging a reservoir chamber with compressed gas from a source and maintaining said gas at a constant volume and pressure.

Use of the method and apparatus of the invention provides for simples particle bombardment having improved economics of operation because the flying block is the only part which is not reused. Replacement of the flying block usually takes only a matter of seconds. The flying blocks are inexpensive and are available with a wide cavity for large particle loads and wide targets, or a narrow cavity for smaller loads and smaller targets. The gun is particularly useful for small targets, such as tissue several millimeters in diameter, in that the particle impact area can be small and the gun can be easily aimed at this area, which is the preferred target for some applications.

Description of the Drawings

Figure 1 is a perspective view of one embodiment of a particle gun apparatus of the invention.

Figure 2 is a cross-sectional view of a particle gun of the invention, with a schematic diagram of the gas propulsion system.

Description of Specific Embodiments

The invention will be illustrated in detail with reference to Figures 1 and 2, which show a preferred embodiment of the invention. In this embodiment the target and a portion of the elongated chamber comprising the opening of the barrel of the gun are located in a vacuum chamber. However, it will be recognized that the invention is not limited to the specific embodiment shown.

A housing (1) is provided that includes a base plate (14) and a target chamber (25), which can be evacuated to give a vacuum and which is located on the support platform or plate (14). A target holder (12) is mounted in the target chamber (25) and is adapted to hold the sample of living cells, tissue, organs, or embryos (12A) for bombardment by one or more microprojectile particles in order to transfer desirable genetic material into the cells. The microprojectile particles are conventionally known in the ar such as those disclosed in U.S. patent 4,945,050, and are conveniently formed from a metal, such as gold. The particles can be of any size known to those skilled in the art of microinjection via particles but preferably have a diameter from about 0.2 μm to about 5.0 μm. The particles are coated or impregnated with the genetic material to be introduced in the manner known in the art

The target cells are held on a target holder plate (12) inverted in a jig (2) which can be assembled into the apparatus at variable, discrete distances from die stripper (3) that acts to stop the forward motion of the flying block (17) used to accelerate the microprojectile particles. The target vacuum chamber (25) is typically maintained at a partial, adjustable vacuum during a shot to minimize air resistance and gas blast A vacuum pump (31) is connected to the target vacuum chamber (25) by a vacuum line (36) and is controlled by an electrical switch (5) or other control device. The vacuum line (36) is connected to a pressure gauge (4) and valves (34 and 35) operable to control the pressure. A vent (32) is provided for venting the target vacuum chamber to the atmosphere subsequent to the bombardment of the biological material so that bombarded material can be removed and a new target inserted.

The barrel of the gun comprises an elongated chamber (6A) having an opening (30) at one end and an empty bore of invariant cross section. The open end is directed toward a stripper holding plate (16) in a partition (13) that divides the target chamber into two parts, the part distal from the barrel of the gun containing the target holder (12) and the part adjacent to the barrel providing access so that the flying block can leave the gun barrel and strike the stripper that stops forward motion of the flying block (forward being in the direction of the target). This stripper has an opening with a smaller cross section than that of the empty bore of the barrel of the gun and of the flying block. The stripper can be located on and directly attached to the bore or proximally a short distance beyond the bore opening between the bore opening and the target In the latter case, which is preferred, the opening of the stripper is located on the principal axis of the bore. In the embodiment of the invention illustrated in Figure 2, the stripper (3) is in the form of a bolt with an axial passageway oriented so that the flying block strikes the end of the bolt and the microprojectiles pass through the axial passageway on their way to the target The physical shape of the stripper (except for the size and orientation of the passageway, as described herein) are immaterial to the invention, but a form that can be firmly but removeably attached to the stripper holding plate is preferred so under the impact of shots by the flying blocks. Threads in the stripper with matching threads in the stripper holding plate (as in the described bolt), separate clamping devices, and numerous other techniques can be used to attach the stripper to the stripper holding plate or other device to which the stripper is attached.

The stripper is located a short distance from the bore opening in this embodiment which represents a preferred embodiment for easy disposal of used flying blocks, which merely fall to the side and are discarded after striking the stripper.

The flying block (17) is the single consumable part of the gun of the invention. It is made of a deformable material, such as a rubber, plastic, deformable metal or the like. One preferred material is a polycarbonate plastic or similar plastic which absorbs kinetic energy upon striking the stripper via plastic deformation rather than fracturing, thereby eliminating debris. A plastic flying block material can also be formed by injection moding, which is an economical mass production method. A preferred shape has the forward (target) end of the flying block formed to provide a shallow depression or cavity (40) that will hold the microprojectile particles. Different shapes can be provided for this depression to allow for different amounts of particles and different patterns of particle dispersion. The back (breech) end of the flying block can have any shape but is preferably configured for easy handling, for example by providing a slot or cavity that fits a breech pin (see discussion below) to allow easy loading of the block into the particle gun.

The stripper (3) can be of any non-deformable material capable of stopping the flying block without significant damage to itself. Obviously, "deformable" and "non-deformable" are relative terms when applied to the flying block and the stripper. However, it will be apparent to one of skill in the art that an object of the invention is to have a flying block that deforms upon striking the stripper while the stripper remains unchanged by the collision, so that no confusion arises out of the use of these terms that are relative to each other. In preferred embodiments the stripper is held at a distance from the end of the barrel in a stripper stage or vacuum chamber partition (13), as previously described. In other embodiments (not shown) the stripper is conveniently a inside-threaded steel cap with an axial passageway smaller than the bore attached to the end of the barrel, which is provided with outside matching threads.

In operating the gun of the invention, the distance from the target to the stripper at which the flying block (17) is stopped can be varied as can be determined by one of skill in the art By way of example, when the target material is suspension-culture cells and the genetic material is DNA or RNA, the flying block (17) is usually stopped at a distance of about 15 mm to about 90 mm, preferably about 25 mm to about 70 mm, from the target Other operating distances can be provided by changing the supporting parts that hold the various working parts, such as the barrel, stripper, and target holder without affecting operation of the particle gun.

The elongated barrel chamber (6A) and flying block (17) can be of any geometrical cross-section (relative to the direction of travel of the flying block).

Usually the shape of the cross-section is circular, but it can be square, rectangular, or any other shape. The flying block fits the barrel chamber in the same manner as the bullet of a gun, namely with sufficient tightness to prevent significant gas leakage but without being too tight so that movement under the influence of the compressed gas is prevented.

The flying clock (17) with the genetic material and particles loaded in the front cavity (40) is introduced into the barrel by carrying the flying block into the breech (6B), typically using forceps; mounting the flying block on a breech bolt pin (6C); sliding the bolt (6E) up into the breech by pushing up in the handle (6D); and rotating the bolt 90 degrees so that the handle engages a slot (6E) which locks the bolt in firing position. The loading mechanism as shown in this embodiment should be familiar, as it is the typical bolt-action loading mechanism of a single shot rifle.

Other breech arrangements to allow loading of the flying block into the barrel of the particle gun can be used if desired. The propulsive force on the flying block is provided by a compressed gas. A compressed gas tank (22) or other source of gas is used to charge a reservoir (23) to the desired pressure. By using a constant volume and pressure of a compressed gas confined in the reservoir (23), the gun provides a controlled, reproducible, adjustable, clean, safe, and inexpensive propulsion source. Single gases, such as helium, nitrogen, argon, or carbon dioxide, or mixtures of gases can be used, giving a very large dynamic range for the expansive force of the gas on the rear of the flying block. Hydrogen, which has the greatest expansive power, can be used but is not recommended for safety reasons, as it is explosive. The pressure of the compressed gas can vary as can be determined by those of skill in the art By way of example, when the compressed gas is helium, the pressure is typically from about 20 psi to about 1500 psi, preferably from about 1000 psi to about 2000 psi.

The gas pressure propulsion source of power for firing the flying block at the target has a wide dynamic range, especially when gases with different densities are used, e.g., helium vs. nitrogen vs. carbon dioxide, because the force available from compressed gases at equal pressures varies as the speed of sound through the gas (the speed of sound is higher as the density decreases). Since bottled gas is pure, it also promotes rapid firing cycle times because there is no cleaning between shots as with the prior art use of gunpowder.

Charging of the reservoir (23) with a constant volume and pressure of gas gives the best possible reproducibility for the propulsion force, surpassing metering valves or rupture disks. This also provides substantially unlimited variation in pressure; the design is therefore more flexible and controllable than the arg discharge or rupture disk particle gun machines. The gun of the invention incorporates minimum dead volumes in the gas pathway such that the minimum volume for space between valve (20) and back of flying block on the breech pin and balances storage and acceleration volumes (i.e., reservoir and barrel size) in order to obtain optimal capture of the propulsion energy. The volume of the barrel is less than the volume of the reservoir. A pressure regulator (10) and electrically operated solenoid valve (21) are located in line (50) between said gas source and said reservoir (23) for controlling the flow of gas into the reservoir. Pressure gauges (9 and 15) show the pressure of the gas supply (22) and reservoir (23), respectively.

A second electrically operated solenoid valve (20) is armed by a first control switch (8) and fired by a second control switch (7). The firing solenoid discharge valve (20) is adapted to discharge the constant volume and pressure gas through line (45) and then through the banjo nut (19) and fitting (18) into the breech (6B) behind the flying block (17) in the embodiment shown. The flying block (17) is thus fired by operation of the firing solenoid discharge valve mechanism. The gas pressure is sufficient to fire the flying block such taht the flying block (17) travels through the bore portion of barrel chamber (6A), carrying the load of DNA-coated particles (17A) in its front cavity (40). The flying block (17) is stopped by the smaller-diameter stripper (3) just past the opening (30) of the bore (6A) in this preferred embodiment. At such time, the particles continue on a straight path to the target cells (12A) while the propulsion gas is diverted to the sides of the barrel (6A). In the embodiment shown, gas is diverted to the sides and away from the target by the stripper and the stripper stage. The deformed flying blocks passage of gas through the suipper; if not diverted this gas could impinge upon and adversely affect the target cells. In embodiments that use a stripper nut attached directly to the bore of the particle gun, openings can be provided in the sides of the bore just behind the back end of the flying block when it strikes the stripper nut so that the propulsive gases can escape to the sides. Release of the compressed gas behind the flying block simplifies removal of the flying block after firing and allows further operation of the gun.

The target holder bearing the biological target material (12A) is held in a moveable plate on the target stage (12). This arrangement permits rapid, predictable, and precise aiming of particles to within a few millimeters distance on the target holder. For convenience, most of the parts of the apparatus are enclosed in a housing (11) to protect them from dust, moisture, and the like.

Reloading of the gun is rapid. The used flying block (17) in this preferred embodiment has left the end of the barrel and is thus merely picked up and discarded, an a new flying block is inserted in the breech (6A). The breech bolt (6C) opens and closes with a single motion. No cleaning of the breech is requred because no combustion occurs in the breech. The target tissue (12A) is aligned, the chamber cover (1) is replaced, and the gas reservoir (23) is recharged. The cycle takes about 15 seconds, as compared to several minutes in the prior art devices.

Maintenance is easy because the parts subject to wear, i.e., the breech bolt (6E), solenoid pistons (20, 21), and stripper (3), can be accessed without fully disassembling the gun and can therefore be replaced in about 15 minutes. The.entire device can be oriented to fire the flying block any desired direction, such as upward, downward, or to the side as is most convenient for example, to simplify holding a variety of target cells in position. The particle gun of the invention has been used to obtain transient expression of introduced nucleic acids, i.e. temporary genetic transformation, in several plant cells and tissues such as tobacco, wheat corn, barley, oat onion, and sorghum, and stable transformation of tobacco, wheat and corn cells.

Example Transformation of Wheat

I. Preparation of the Genetic Constructs Used in the Bombardment

PSOG IO

This β-Glucuronidase (GUS) expression vector was derived from plasmid pBl 121, purchased from Cloneteck Laboratories, Palo Alto, Califormia. Intron 6 of the maize Adhl gene was amplified by PCR from plasmid pB428, described in "Bennetzen et al., Proc. Natl. Acad. Scl, USA 81:4125-4128 (1987)", using oligonucleotide primers SON0003 and SON0004.

SON0003: 5'-CTCGGATCCAGCAGATTCGAAGAAGGTACAG-3' SON0004: 5'-ACGGGATCCAACπCCTAGCTGAAAAATGGG-3'

The PCR reaction product was digested with restriction endonuclease BamHl, cleaving the BamHl site added on the 5' end of each PCR primer. The resulting DNA fragment was purified on an agarose gel and ligated into the BamHl site of pB1121, which is between the CaMV 35S promoter and the CUS gene. The ligated DNA was transformed into E.coli and clones with the Adhl intron 6 in the same orientation as the GUS gene were identified by restriction digest

PSOG 19

This dihydrofolate reductase (DHFR) expression vector was derived by fusing the 35S promoter and Adhl intron 6 of pSOG 10 to the DHFR gene from plasmid pHCO, described in "Bourouis and Jarry, EMBO J.2: 1099-1104 (1983)". The 35S promoter and Adhl intron 6 were produced by PCR amplification of the fragment from pSOG 10 using primers SON0031 and SON0010. SON0031: 5'-CATGAGGGACTGACCACCCGGGGATC-3' SON0010: 5'-AGCGGATAACAATTTCACACAGGA-3'

The resulting fragment was digested with restriction endonucleases Pstl and BspHl and purified on an agarose gel.

The DHFR coding region was produced by PCR amplification of pHCO using primers SON0016 and SON0017.

SON0016: 5'-GCTACCNrGGCCACATAGAACACC-3' SON0017: 5'-CGAGAGCTCCiCACπCAACCπG-3'

The resulting fragment was digested with restriction endonucleases Nsol and Sacl and purified on an agarose gel.

The two fragments described above were ligated into a vector fragment prepared from pB1121 by digestion with restriction endonucleases Pstl and Sacl and purification of die 3 kb fragment containing the Nos terminator region and pUC19 region of pB1121 on an agarose gel. This three way ligation fused the 35S promoter- Adhl intron 6-DHFR gene-Nos terminator in correct order and orientation for functional expression in plants.

PSOG 30

This GUS expression vector was derived from pSOG 10 by the insertion of the maize chlorotic mottle virus (MCMV) leader, described in "Lommel et al., Virology 181: 382-385 (1991)", into the 35S-GUS gene non-translated leader by a three way ligation.

Both strands of the 17 bp MCMV capsid protein leader sequence plus appropriate restriction endonuclease sights were syntiiesized and annealed. The resulting double stranded fragment was degested with BamHl and Ncol and purified on an acrylamide gel.

The GUS gene coding region was amplified by PCR using primers SON0039 and SON0041 and pB1121 as a template.

SON0039: 5'-CGACATGGTACGTCCTGTAGAAACCCACA-3' SON0041 : 5'-ATCGCAAGACCGGCAACAGGATTC-3'

These primers added an Ncol site to the 5' end of GUS and a Sacl site to the 3' end of GUS. The resulting fragment was digested with restriction endonucleases Ncol and Sacl and purified on an agarose gel.

The GUS gene eas removed from the plasmid pSOG 10 by digestion with restriction endonuclease Sacl and partial digestion with restriction endonuclease BamHl. The resulting vector, which has a BamHl site and a Sacl site in which to reinsert a coding region behind the 35S promoter-Adhl intron 6, was purified on an agarose gel.

The three fragments described above were ligated in a three way ligation to produce a gene fusion with the structure: 35S promoter-Adhl intron 6-MCMV leader-GUS-Nos terminator, all in the pUC 19 vector backbone.

PSOG 35

The DHFR selectable marker vector is identical to pSOG 19, except that the MCMV leader is inserted in the non-translated leader of the DHFR gene to enhance translation. It was created in two steps. First the GUS coding region in pSOG 32, a vector identical to pSOG 30 except that it contains a modified Adh promoter rather than 35S, was replaced with DHFR coding region from pSOG 19 by excising the GUS with Ncol and Sacl and ligating in the DHFR as an Ncol-Sacl fragment This resulting in vector pSOG 33 which has the gene structure Adh promoter-Adhl intron 6-MCMV leader-DHFR coding region-Nos terminator, with a Bglll site between the promoter and Intron and a Sacl site between the coding region and the terminator. The Bglll-Sacl fragment was isolated by restriction endonuclease digestion and agarose gel purification, and ligated into the BamHl and Sacl sites of pSOG 30, replacing the Adhl iπtronZ6-MCMV leader-GUS coding region of pSOG30 with the Adhl intronZ6-MCMV leader-DHFR coding region of pSOG33.

II. Cell Culture Maintenance

Callus cultures are maintained on IMS medium, as described for example by "Murashige, T. and Skoog F., 1962, Physiol. Plant 15, 473 - 497" (MS salts, vitamins, iron, 3% sucrose, 0.7% agar, 1 mg/liter 2,4-D). The suitable callus cultures comprise among others Type π callus (a friable and embryogenic type of callus) obtained from "shoot-competent" cell cultures as described in "W. Wang and H. Nguyen, 1990, Plant Cell Reports 8639-642") after recurrent subculture and visual selection. They are subcultured every two weeks and are kept in the dark at 26°C. Suspension cultures are maintained in IMS liquid medium and are subcultured twice weekly. They are kept in the dark at 26°C and shaken at ~125 rpm.

in. Cell Preparation for Bombardment The cells are given a plasmolysis treatment before bombardment. Packed cell volume is measured and cells are diluted in IMS liquid medium with added osmoticum: 0.4M sorbitol for suspension cells and 0.6M sorbitol for callus cells. Cells are diluted such that the final packed cell volume per target is 1/20 ml for a fine suspension and 1/10 ml for callus. Diluted cells are placed in a 250 ml flask containing a stir bar and are stirred for a minimum of 30 minutes, up to a few hours. To plate the cells, 2 ml is withdrawn from the flask and pipetted into the top of a vacuum flask onto which a Whatman 2.5 cm GFA filter has been placed. The vacuum is applied until the cells are dried onto the filter, the filters are placed on 60x15 mm petri plates containing 5 ml of solid post bombardment plasmolysis medium: IMS containing 0.2M sorbitol for suspension cells, or 0.4M sorbitol for callus cells. Two filters are plated on each dish.

IV. Particle Preparation

Gold particles (1.0 micron; from Bio-Rad) are washed by aliquoting into a microfuge tube, adding ~1 ml 100% ethanol, vortexing, spinning down, removing die supernatant, and repeating twice with sterile water. After the final wash, as much water is removed as possible and polylysine solution (0.02% polylysine + 15 mM ammonium acetate) is added to completely immerse the particles. The particles are vortexed, spun, and the supernatant removed. The particles are allowed to dry overnight in a laminar flow hoodor for 30 minutes under a gentle nitrogen stream.

For a "full" particle preparation, weigh out 10 mg particles and place in sterile microfuge tube containing a stir bar. Add 100 μl (1 μg/μl) DNA (according to step I), vortex, add 10 μl 100 mM Na₂HPO₄, vortex, add 10 μl 100 mM CaCl₂, vortex, add 380 μl 100% ethanol, vortex. Stir suspension vigorously on stir plate while pipetting 3 μl onto each plastic flier (projectile). Allow particles to dry onto fliers for at least 15 minutes before bombarding.

V. Bombarding Cell Cultures

Invert the petri plate containing the cell filters onto the platform on top of the stage, centered over the particle flight opening. Place the clear lid over the top of the platform. Place a microprojectile onto the breech pin and close the breech. Push the "arm" button ro fill the reservoir with the appropriate amount of helium gas (usually 1800 - 1900 psi). Pull the vacuum on the chamber to ~27 mm. Turn off the vacuum, and push the "arm" and "fire" buttons. Move the "arm" button to the "off position. Each filter is usually shot twice.

VI. Post-bombardment Culture and Selection

After bombardment the cells are kept in the dark overnight The next day, filters are removed from plasmolysis medium and places on IMS medium. Selection is applied 7 - 10 days post-bombardment for suspension cells and after 14 days for callus cells. Cells are scraped off the filters and spread onto the surface of plates containing IMS plus 2 mg/liter methotrexate. (Transformants may be obtained by initially selecting at 4 mg/liter methotrexate also.) Plates are incubated in the dark for several weeks. Resistant colonies that arise after a few weeks are transferred to IMS + 4 mg/1 methotrexate. Colonies that continue to proliferate for about 3 - 4 weeks are then transferred to "0.5MS" maintenance medium: MS salts, vitamins, iron, 3% sucrose, 0.7% agar, 0.5 mg/liter 2,4-D. Tissue is subcultured onto tiiis medium biweekly until embryogenic structures appear or tissue seems suitable for regeneration.

VB Regeneration

Tissue is transferred to MS medium containing either 3 mg/liter BAP or 1 mg/liter NAA + 5 mg/liter GA, and plates are moved to the light After 2 - 4 weeks, tissue is transferred to MS medium without hormones. Shoots that appear are placed in Magenta boxes containing either MS medium without hormones or MS medium with 0.5 mg/liter NAA. When sufficient root and shoot growth has occurred, plantlets are transferred to soil and placed in a phytotron.

VHL Transform Analysis

About 20 mg callus tissue is used for PCR analysis. DNA is extracted using a quick phenol chloroform:isoamyl alcohol method and 2 μl is used per reaction. Primers have been designed to amplify the region from the 5' end of die Adh gene to die 3' end of die DHFR gene.

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

Claims

What is claimed is:

1. A particle gun for the introduction of caπier particles carrying genetic material into intact cells, comprising: an elongated chamber having a principal axis and an opening located at one end of said principal axis, said chamber having an empty bore portion of invariant cross section centered on said principal axis; a cell carrier target holder located outside said chamber on said principal axis and adjacent said opening; stripping means located on said principal axis between said target holder and said bore opening, said stripping means having an opening therethrough located on said axis with a cross section smaller than mat of said bore portion; means for introducing a deformable flying block into said chamber, wherein said flying block is adapted to move along said principal axis in said bore portion while substantially blocking passage of compressed gas past said flying block, said flying block being too large to enter said opening in said stripper means, wherein a surface of said flying block proximal to said opening when said flying block is in said bore is adapted to hold said carrier particles and wherein said flying block is moveable by a compressed gas yet stoppable on contact of said flying block witii said stripping means; a compressed gas reservoir having a controllable volume and pressure; and means for introducing said compressed gas from said reservoir behind said flying block relative to said opening; whereby when said carrier particles are loaded onto said flying block and cells are present on a target in said target holder, entry of compressed gas into said chamber causes said flying block to travel along said axis until said flying block is stopped by said stripping means thereby releasing said carrier particles which continue to travel through said stripping means opening into said cells.

2. The particle gun of claim 1, wherein said target holder and a portion of said elongated chamber encompassing said bore opening are located in a vacuum chamber.

3. The particle gun of claim 1, wherein said means for introducing said flying block into said chamber comprises a breech portion located on said principal axis distal to said opening.

4. The particle gun of claim 1, wherein said stripper means is mounted proximal to but outside said bore opening.

5. The particle gun of claim 1, wherein said stripper means is mounted on said elongated chamber at said bore opening.

6. The particle gun of claim 1, wherein said bore and said flying block are cylindrical.

7. The particle gun of claim 1, wherein said stripper means is located a short distance from said bore opening, said distance being longer than a length of said flying block, as taken along said principal axis.

8. The particle gun of claim 7, wherein along said distance between said bore opening and said suipper means mere extends an empty space, adapted for a disposal of said flying block after striking the stripper means.

9. A method for the introduction of genetic material into intact cells, wherein said genetic material, which one desires to have introduced into said intact cells, is prepared, wherein ballistic means are used for accelerating said genetic material at a velocity sufficient to enter into said intact cells and thereby transform them, without damaging said cells, and wherein shoots and whole plants are generated from said transformed cells.

10. The method of claim 9, wherein wherein said genetic material is carried on carrier particles; wherein said ballistic means is a particle gun having said carrier particles loaded onto a flying block in an elongated chamber having a principal axis and an opening located at one end of said principal axis, said chamber having an empty bore portion of invariant cross section centered on said principal axis; wherein said flying block is adapted to move along said principal axis in said bore portion and strike an obstruction, whereby momentum imparted to said carrier particles causes said carrier particles to continue to move forward and inject said genetic material into said intact cells, which are located on said axis outside said chamber, and wherein a compressed gas is injected from a reservoir, where it is maintained at a constant volume and pressure, into said chamber behind said flying block and distally to said flying block in said chamber relative to said opening.

11. The method of claim 10, wherein said compressed gas is nitrogen or helium.

12. The method of claim 11, wherein said compressed gas is helium.

13. The method of claim 10, wherein said flying block is stopped by a stripper means located a distance of from about 15 mm to about 90 mm from said intact cells.

14. The method of claim 13, wherein after striking said stripper means said flying block is discarded through an empty space provided between said bore opening and said stripper means, said empty space being located a short distance from said opening, which distance is chosen longer than a length of said flying block, as taken along said principal axis.

15. The method of claim 10, wherein a target holder and a portion of said elongated chamber encompassing said bore opening are located in a vacuum chamber.

16. The method of claim 10, wherein compressed gas is supplied to said chamber at a pressure of from about 20 psi to about 2500 psi.

17. The method of claim 9, wherein said intact cells are taken from the group of plant cells.

18. The method of claim 17, wherein as said plant cells are living cells chosen from tobacco, wheat corn, barley, oat onion or sorghum plants and further wherein said plant cells are temporarily transformed.

19. The method of claim 17, wherein as said plant cells living cells from tobaco, wheat corn or barley are chosen and further wherein said plant cells are permanently transformed.

20. The method of claim 19, wherein said plant cells are wheat cells.

21. Wheat plants transformed according to claim 20.