CA2062178A1 - Aerosol beam microinjector - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
The present invention uses aerosol beam technology to accelerate either wet or dry aerosol particles to speeds enabling the particles to penetrate living cells. Aerosol particles suspended in an inert gas are accelerated to a very high velocity during the jet expansion of the gas as it passes from a region of higher gas pressure to a region of lower has pressure through a small orifice. The accelerated particles are positioned to impact a preferred target, for example, a plant or animal cell or bacterial culture. When the droplets include DNA or other macromolecules, the macromolecules are introduced into the cells. The particles are constructed as droplets of a sufficiently small size so that the cells survive the penetration. Once introduced into the target cell the macromolecules can elicit biological effects. Because the method of introduction is a physical one, the biological barriers that restrict the application of other DNA transfer methods to a few plant species and a few cell types are not present. In addition, the method and apparatus of the present invention permit the treatment of a large number of cells in the course of any single treatment.
Thus the inventive method and apparatus should be applicable to a wide range of plant species and cell types that have proved in the past to be quite impervious to standard methods of genetic engineering.

Description

UOgl/00915 PCT~US90/0~3 -AE~OSO~ BEAM MICROI~ECTOR

The presen~ invention relates to the introduction of exogenous material into a living cell, tissue, or species, and more particularly, to apparatus and methods for lS microinjecting exogenous materials into a living cell, tissue, or species.

The genetic transfor~ation of cells using the techniques of genetic engineering has grown dramatically over the last few years. Generally, genetic engineering involves the introduction of foreign DNA into a host cell to change or "transform" the host cell. When successfully done, the genes carried by the foreign DNA are expressed in the host cell, and thus, the last cell is transformed.
Genetically transformed cells, tissues, or species are of great commercial value in research, agriculture, and medicine. For example, human insulin is presently being produced~by~bacteria which have been genetically èngineered. Human insulin is now available in co~mercial quantities, and ha~ benefitted milllons o~ diabetics throughout the world. In addit~on, several plant speci~
have been gene~ically engineered to produce more crop, becom- more resistant to disea~e, and grow in less hospitabl- climates and soils.

Because of the commercial and social importance of genetic engineering, a plurality of methods ~or transferrlng for-ign genes into a recipient cell have been .:

developed for both procaryotic and eucaryotic cells.
These gene transfer techniques are routinely used in laboratories throughout the world to genetically transform living cells, tissues and species. However, despite the existence of these diverse transformaticn techniques, the effective stable transfer of genes remains an obstacle in many fields of research. One technique for gene transfer i8 cell fusion. Cell fusion has been applied to both animal and plant cell cultures. Cell fusion techniques integrate the genetic information of two different cells in one. In plant cell cultures, cell walls are dissolved and the naked protoplasts are fused together, forming a new cell. The descendants of the new ~used cell express certain characteristics of both the original (parent) cells. However, researchers have no means of predicting what characteristics will be expressed in the progeny.
Thus, the results obtained using cell fusion techniques are unpredictable and are generally not reproducible.

In animal cell cultures, hybridomas are the cell fusion product of antibody-secreting cells and "$mmortal"
cells (myelomas). Hybridomas are used in medicine and research to produce monoclonal antibodies. Hybridomas, like the plant cell fusion products, may react unpredictably, and are expensive to produce and maintain.

Electroporatlon is another general method for introducing ~oreign DNA into cells. ThiC procedure involves the exposure of a suspension of cells and fragments of foreign DNA to a pulsed high-voltage electrical discharge. This electrical discharge creates holes or pores in the cell membrane through which DNA
fragments dif~use. Upon cessation of the electrical discharge, the pores in the cell membrane close, capturing ; 35 any DNA fragments which have diffused into the cell.
~ However, a major drawback in using electroporation to W~) 91/0091~ . PCIIUS90/03663 2062~78 introduce DNA into living cells is the necessity prior to a successful transformation, to determine empirically optimum values for a wide variety of parameters for each cell line in guestion. Thes~ parameters include voltage, capacitance, pulse time composition and resistance of electroporation medium, state of cell growth prior to electroporation, concentration of DNA, temperature, and cell density. Also, pr~sent methods for applying electroporation to cells with-walls, such as plant cells, require removing the walls prior to electroporation.
~hese protoplasts must then regen~rate their walls b~fore they can divide and be regenerated into intact plants.
Since not many plants can yet be regenerated from protoplasts, this requirement limits applicability of electroporation.

Viral infection is also an efficient means of transferring foreign DNA into some cells. However, viruses are difficult to work with, and since a particular virus will only infect certain cells, the technique is not applicable to the broad spectrum of living cells, tissue, or species. Further, some virus may in fact be hazardous to the technicians working with them or to the environment.
The most common and effective method for transferring genes into plant cells involves conjugal transfer from living bacteria, primarily AarQbacteriu~ ~umefacie~t and Aarob~cterium rhiz~qenes. The DNA to be transferred is first engineered into plasmids derived from the bacterial ~i plasmid, and thQ bacteria are then incubated in culture with the recipient plant cells. The cultured, transformed cells must then be regenerated into genetically homogeneous plants. This method is limited to plants susceptible to Aarokacterium infection (principally dicots) that can be regenerated from cultures.

Still another technique for gene transfer is micropipette microinjection. In micropipette microinjection, DNA or other exogenous materials, are stably introduced into a living cell ffl microinjecting the material directing into the nuclei of the cell.
Typically, a glass micropipette having a diameter of from about 0.1 to about 0.5 microns is inserted dlrectly into the nucleus of the cell. Through the bore of the micropipette exogenous material, such as DNA, is in~ected.
An exparienced, skilled technician can in~ect from about 500 to about 1000 cells per hour. Presently, however, ~icropipette microinjection requires some fairly sophisticated and expensive equipment, such as, a micropipette puller for maXing the needles, and a micromanipulator to position the needles correctly for injection. Moreover, extensive practice and training is needed for a technician to master this tedious technique.
Micropipette microin;ection is an effective method of gene transfer; however, because of the relatively small number of cells that may be trans~ormed in any one experiment, the cost of the elaborate equipment necessary, and the level of skill needed to perform the procedure, microinjection using micropipettes i5 not a mqthod of gene transfer availa~le to the v~st ma~ority of researchers in the field.

Altbough a number Or gane trans~or techniques are ~vailable~ pr-sently, no prior technique provides a safe, ~ast, e~fe¢tive, lnexpensive, and reproducible method for ganetically transforming a broad spectrum of living material regardless of cell, tissue or species type.
Pre~ent methods are either expensive, slow, or yield results which ar- not reproduc~ble. Moreover, some 3s metbods, sucb as the use of infectious agents, may even be hazardous to the researcher or the environment.

-' ' ` ' W091/009l~ PCTlUS90/03663 ~5~ 2062178 Accordingly, a new apparatus and method for trans~ormingcells, t~ssue, or species is needed.

Advantages of the invention will ~ecome apparent upon S reading the following detailed description and upon reference to the drawings in which:

FIG. 1 is a schematic illustration of the aerosol beam microinjector;
Fig. 2 is a schematic illustration o~ an aerosol beam microinjector including a dry~ng tube and means ~or providing sheath flow; and Fig. 3 is a schematic illustration of an aerosol beam microinjector addi~ionally including means for adjusting the diameter of the injected droplet.

The present invention is directed to an apparatus and methods for the introduction of exogenous materials~ for example foreign DNA, into a variety of recipient cells.
It is believed that the pres~nt invention should have a profound effect upon the variety of plants, and animals, that will become amenable to direct genetic manipulation.
The present invention uses aerosol beam technology to accelerate either wet or dry aerosol particles to speeds enabling the particles to penetrate living cells upon impact therswith. Aerosol pArtioles suspended in a inert gas can be accelerated to a very high velocity during the ~et expansion of the gas as it pa~5es from a region of higher gas pressure to a region of lower gas pressure through a small orifice. This acceleration process has been well-studied and usQd for many years in the field of aerosol physics. The accelerated droplets may be ~ positioned to impact a preferred target, for example, a ..
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2062~78 plant or animal cell. When the particles include DNA or other macromolecules, the ~acromolecules are introduced into the cells. The droplets may be constructed as droplets of a sufficiently small size so that the cells survive the penetration. Once introduced into the target cell the macromolecules can el$cit biological effects.
This effect will depend on the specific properties of the macro~olecules and the nature o~ the recipi~nt cells. For example, DNA may be introduced into the target cell, thereby altering the genetic make-up of the cell. Because the method of introduction is a physical one, t~e biological barriers that restrict the application of other DNA transf0r methods to a few plant species and a few cell types are not present. The size, kinetic energy and beam intensity of the aerosol stream can be precisely controlled over a wide range, and hence may be successful in penetrating and transforming a wide variety of cell types. In addition, the use of a continuous aerosol beam will permit the treatment of a large number of cells in the course of any single treatment. Thus, the inventive method should be applicable to a wide range of plant species and cell types that have proved in the past to be quite impervious to standard methods of genetic engineering. According to a preferred embodiment, the apparatus and methods of the present invention are utilized to produce a transformed line of plants. Aerosol part~¢les comprising Qxog~nous genetic material are produced and acceleratsd to a speed enabling them to p~netrate and enter into a living plant cell upon impact therewith. The cells are thereafter cultured to grow a plant. The progeny of the plant are subsequently screened using techniques well known to one skilled in the art of plant qenetics, for transrormed progeny.

According to a preferred embodiment, the apparatus and methods of the present invention are utilized to produce transformed maize plants. Cells from an embryogenic culture of an alcohol dehydrogenese-deficient ~adh ) recipient plant ar~ preferably plated on an agar surface of osmotically supportive medium. They would then be placed in the chamber on the target positioning device for treatment with aerosol carrying a mild type ahd gene on a plasmid. The apparatus shown in FIG. 3 is preferably used. The DNA plasmid would be ad~usted to 3mMMyCl~ and rapidly mixed with 100% ethanol to yield 95% final ethanol concentration. The concentration of DNA would ~e such that each droplet of primary aerosol g~nerated in the nebulizer contains an average o~ 2-3 plasmid molecules (about 10~3 molecule per ml of final ethanol solutions).
The primary aerosol, generated in the nebulizer would then be dried and the ethanol vapor trapped in the d~fusion dryer. The DNA aerosol stream would be humidified with 5xlO 16 g water vapor per primary aerosol particles, and the mixture cooled to 0c. to generate a 0.5 - l.0 mm diameter uniform size aerosol droplets. These would be accelerated by the expansion of the N2 inert gas through a 2S0 mm orifice to strike the target cells l.5 cm away from tha orifice. The injected cells would then be cultured and tested by cytological staining for the presence of adh activity in the cells. An appropriate number of cells would be grown into plants and for the tested for the pr~sence of the transforming DNA and for function of the adh gene.

A further preferred embodim~nt of the present invention utilizes the apparatus and methods o~ the present invention genetic transformation of maize pollen.
Pollen from a recipient variety deficient in adh would be plated on osmotically supportive agar m~dium and placed in the device shown in FIG. 3. Transforming DNA would be 3S for~ed into a primary aerosol of 95% ethanol a~ described for embryogenic cell transformation, and tben dried as set forth above. In this case, the aerosol stream would be humidified with perfluorocarbon vapor (3M or Air Products) at a density of 8xlO 12 g per primary aerosol droplet.
Condensation at 0C would then generate 2 micron uniform diameter droplets. These would be accelerated with the inert gas expansion to impact the pollen grains. After 1 hr recovery period, the pollen would be transferred to silXs of 3~h deficient corn plants. ~he 9eeds obtained would be planted and trans~ormed plants recognized by the presence of adh-stainable pollen in samples ~rom the mature plants.

~ erosol particles are formed by aerosolizing solutions containing the macromol~cule. Preferably, excess solvent is subsequently evaporated from the aerosol particles to reduce projectile size, thus increasing the rate of survival in the impacted target cells. When the aerosol particles are constructed as droplets the density of the final droplets is important, since less dense droplets may cause less damage to some target cells when they penetrate, while, on the oth~r hand, more dense droplets may be required to penetrate certain target cells. Accordingly, the present invention provides means to modify both the size and density of the droplets as required for penetrating different cells. According to onQ preferred embodi~ent, the density o~ the droplets i8 from about 0.8 to about 8 gram/cc, and mo8t pre~erably about 2 grams/cc. The density is ~odified by the addition of solutes, for example colloidal gold, to the solution prior to forming an aerosol or by using solvents, such as perfluorocarbons, that have different densities. Further, the apparatus of the present invention may be modified to include means for removing excess solvent, thus reducing droplet size, or adding additional solvent to the droplet, thus increasing the size of the droplet.

WO91/00915 PCT/US90/0~3 Figs. 1, 2 and 3, illustrate several embodiments of an aerosol beam microinjector of the present invention.
Each Fig. will be discussed in detail when appropriate~
otherwise the following discussion is applicable to each FIG. Compressed inert gas is filtered as it passes through a tubing l into the inlet 3 of a nebuliser 5 disbursing aerosol particles (a suspension of solvent and solute) into the inert gas to form an a~rosol. ~he source of the compressed gas 6 preferably provides a gas pressure of from about lO to about lO0 psi at the inlet 3.
However, the compressed gas most preferably provides about 30 psi at the inlet 3. Preferably, the projectiles (droplets of solvent and suspended particulate matter) have mass mean diameters of from about O.l to about 2 microns. The present inventor has determined that projectiles having diameters outside of this range are not preferable in the practice of the present invention.
Proj~éctiles having a diameter of greater than about 2 microns were found to cause an unacceptable level of cell death following impact, while pro~ectiles having a diameter of below about O.l microns were unable to efficiently penetrate into the target cell.

The aerosol exits the nebulizer 5 through an outlet 7. The present inventor has determined that it is preferable to dissipate the pressure of the compressed gas prior to the acceleration of the aerosol. This is accomplished, in one preferred embodiment, by venting the aerosol to the outside atmosphere. As shown in Fig. l, 2, and 3, the outlet 7 and the inlst 9 of tubing ll do not abut each other, but are rather, positioned in a close proximity to each other. This arrangement allows the pressure of the compressed gas to dissipate, while further allowing the aerosol to enter inlet 9 of tubing ll.
According to a further preferred embodiment not shown in the FIGS. the aerosol is discharged from the outlet 7 into a non-restrictive enclosure, such as a non-reactive plastic bag. once the gas pressure fro~ the compressed gas has been alleviated, a portion of the aerosol is drawn into the inlet 9 of a tu~ing 11. The aerosol is drawn into the tubing 11 by a negative or low gas pressure (a gas pressure below that of the surrounding atmosphere).
This negative pressure is preferably about 1 atmosphere and is crea~ed within the tubing 11 by a high-volume vacuum pump 13 in communication with an air-tight vacuum housing 15 which is functional~y connected to the outlet 16 of the tubing 11. I~ the aerosol is being discharged into the atmosphere, the inlet 9 of the tubing 11 is preferably positioned in a relatively close proximity to the nebulizer outlet 7. Preferably, this distance is about one-half inch.

Referring specifically to Fig. 1, Fig. 1 shows the tubing 11 entering the housing 15. Fig. 1 shows the tubing outlet 16 including a nozzle 17 through which ths iner~ gas and the aerosol eventually passes through to reach the vacuum housing 15.

Returning, generally, to Figs. 1, 2, and 3, the pressure withi~ the tubing 11 and correspondingly within the vacuum housing 15 may be regulated by the pumping speQd of the pump 13, the flow resistance caused by the nozzle 17, or the evaporation of solvent ~rom samples within the vacuum housing 15. The aerosol is drawn thr~ugh the length o~ tubing 11 to the nozzle 17. The nozzle 17 preferably ha~ an aperture diameter of from 100 to 300 microns. The present inventor has determined that the most preferred aperture dia~eter of nozzle 17 is about ; 200 microns. The aerosol is thereafter drawn through nozzle 17 and into the vacuum housing lS.

WO91/009l5 PCT/US90/03663 2062~78 The passage of the aerosol from the relatively high gas pressure area within tubing 11 into the relatively low gas pressure area within the vacuu~ housing 15 causes the inert gas to dramatically expand. The rapid expansion of the inert gas through the nozzle 17 causes the aerosol particles to accelerate. For example, when nitrogen is used as the inert gas, it has been determined that the aerosol particles can be accelerated up to speed~ of from about 400 to about 800 meters/second. Further, i~ helium is used as the inert gas, the aerosol particleæ may be accelerated up to speeds of about 2000 meterslsecond. The vacuum housin~ ~5 is continuously evacuated by the pump 13 to remove tbe inert gas and keep the pressura in the housing below the gas pressure in the tubing 11.
According to one preferred embodiment, the gas pressure in housing lS is from about 0.01 to about 0.05 atmospheres, and most preferably about 0.01 atmospheres.

When the inert gas is expanded and the aerosol is accelerated, an aerosol beam is generated. In the vacuum housing 15 the inert gas portion o~ the aerosol is expanded and remo~ed and accelerated aerosol particles follow straight line tra~ectories. Aerosol beams are thus composed of accelerated isolated particles and droplets (hereinafter referred to as simply projectiles) moving on well defined, straight line trajectories at speeds up to 2000 meters/seconds.

Referring now to F~g. 2, Flg. 2 illustratos an aerosol beam microinjector including means for msnipulating the size of the pro~ectiles, and means for focusing the aerosol flow through the nozzle 17. As the aerosol is drawn through tubing 11 it is drawn into a drying tube 23. Drying tube 23 contains a desiccant which traps solvent evaporated from the surface of the solute, thus decreasing the diameter of the projectile. The flow 20~2~78 rate of the aerosol, the length o the drying tube, the solvent used, and the desiccant contained in the drying tube, may all be modiied to remove varying amounts of solvent from the aerosol. According to one preferred S embodiment, the drying tube 23 has an internal diameter of about one-half inch and an effective length of a~out 60 cm. ~he drying tube 23 contain~ silica gel as a desiccant. The silica gel surrounds a central channel formed by a cylinder o~ 20 mesh stainless steel screen.
Com~erc~al drying tubes ~imilar to the drying tube described above and use~ul in the practice of the present invention are available ~rom TSI, Inc., Model 3062 Diffusion Dryer.

Once treated, the aerosol continues through tubing 11. The aerosol is discharged from outlet 7 of tubing 11 and into the precise center of a laminar flow of dry, ~iltered inert gas. ~he aerosol i9 thus entrained in the center of a gas stream moving up to and through the nozzle 17. This is preferable ~ecaus~ it increases the average velocity o~ the projectiles, focuses the aerosol beam, and prevents nozzle clogging. When a gas flows through a nozzle 17, the ~elocity profile is never constant over the entire cross section. The particles nearest the nozzle wall will have velocities substantially less than particles traveling in the center of the beam. Further, the radial expansion of the carrier gas will qause the aerosol ~eam to expand radially outward. Particles near the beam center are not influenced by this radial expansion but particles at increasing radii obtain an increasing radial velocity component. Accordingly, Fig. 2 illustrate~ an aerosol beam microin~ector which includes means or reducing the radial expansion of the aerosol beam and maintaining a substantially uniform velocity through the cross section of the beam.

WO91/~0915 PCT/US90/0~3 Fig. 2 shows a piping l9 drawing filtQred inert gas into its length and transporting that gas to the inlet 2l of the sheath flow piping 24. T~e inert qas, which accounts for the sheath flow, enters the pi ping 19 with no positive pressure and is drawn from a non-restrictive reservoir of inert gas or from the outside atmosphere when the inert gas is released fro~ a source 26 in the immediate area of the inlet 25 of piping l9, as shown in Fig. 2. Tha aerosol is discharged from tub~ng ll into the center of the sheath flow piping 24. The aerosol i8 entrained in the laminar ~low o~ the inert gas in the she~th flow piping 24. The laminar ~low focu~es the aerosol through the center of the nozzle 17, thus allowing the aerosol to maintain a substantially uniform velocity across its cross section. The laminar flow also r~duces beam spreading so that a more focused beam is obtained.
According to one preferred e~bodiment the laminar flow accounts for 50% of the flow through the nozzle 17.

Referring to Fig. 3, Fig. 3 illustrates an aerosol beam microinjector having additional means to ~ontrol particle size. As in the apparatus illustrated in Fig. 2, the apparatus of Fig. 3 includes a drying tube 23.
However, in the apparatus of Fig. 3 the drying tube removes substantially all the solvent from the aerosol.
Thus, the aerosol exiting the drying tube 23 through the tubing ll is comprised substantially o~ dry solute. The solute is discharged from the tubing ll into a channel 27 o~ a gas reheater 29. In addition to the dry solute solvent, solvent saturated cArrier gas ~ 8 dischargQd into the channel 27.

Tha solvent saturated inert gas is produced in the vapor saturator 3l and is transported through the piping l9 to the channel 27 of the reheater 29. F~ltered dry inert gas is drawn from 3 source 26 into the piping l9, as . . ~
.. ' . `, . ~ ~
-. ' ~ ' -discussed for the apparatus of Flg. 2. The inert gas thereafter enters the vapor saturator 31, wherein solvent vapor is added to the inert gas to the point of saturation. The vapor saturator 11 is preferably constructed to contain solvent moistened ~ibrous material forming a central channel 33. Surrounding the solvent-moistened material is preferably an aluminum block 28 hoated to a desired temperature with resistive heaters 30.
Preferably, this temperature is ~rom about 50 to about 100 C. The heat causes the solvent moistened material to release solvent vapor which is entrained by the inert gas.
Although this i5 a preferred construction for the vapor saturator 31, other means to saturate the inert gas with solvent vapor may be utilized in the practice of the present invention.

~ he solvent-saturated inert gas is discharged from the vapor saturator 31 intQ piping 20 which co3municates with channel 27 of the reheater 29. The dry aerosol is discharged from tubing 11 also into the reheater 29. The reheater 29 heats th~ dry aerosol and the solvent-saturated inert gas to a constant temperature. This facilitates the dif~usion and mixing of the vapor-saturated inert gas and the dry aerosol particles.
Preferably, the resulting mixture is heated to about 50 C.

From the channsl 27 o~ the reheater 29 the solvent-saturated inert gas and the dry aerosol particle~ mov~ to a vapor condenser 35. The vapor condenser 35 is preferably constructed as having a central channel 37 wherein by reducing the temperature, the solvent vapor is condensed onto the aerosol particles. Depending on the temperature within the channel 37, the amount o~ solvent condensed may be controlled. Thus, the projectile size may be precisely controlled. According to one embodiment, the vapor condenser 35 includes Lapeltier Coolers, WO91/00915 l5 rcT/us9o~o3663 obtained from Marlow Industries. The vapor con~enser outlet 39 includes the nozzle 17, and is positioned within the vacuum housing 15.

Referring again to Figs. 1, 2, and 3, placed in the path of these projectiles is the tarqet support platform 41. The target support platform 41 is a substantially horizontal surface capable of supporting target cells thereon and being preferably movaable along the X, Y and Z
axis. According to the most preferred embodiment, the target support platform 19 i8 provided with, supported by, and affixed to a motorized positioning m~mber which enables it to be positioned either closer or farther away from the nozzle 17. Through experimentation, the inventor has determined that the target support platform 41 is preferably positioned about 1.5 cm from the nozzle 17.
However, this distance may be varied depending on the specific application. For example, it has been determined that the greater the distance the target support platform 41 is from the nozzle 17 the slower the impact speed of the projectiles. This is due to the background pressure in the vacuum housing 15 reducing the speed of the projectiles as they travel through the housing 15.
According to the most preferred embodiment, the target support platform 41 is rotated and is moved by the positioning member along one linear axis 43. This allows for a biological sample placed on the target support platform 41 to be impacted throughout its entirety by pro~ectiles. The presQnt inventors had determined that an ef~ective rate of ro,tation about axis 43 i8 40 rpm and that an effective rate of simultaneous linear advance along axis 43 is 333 microns per revolution. This allows the entirety of a biological sample of approximately 5x7 centimeters to be impacted (hereinafter referred to as scanned) in three to four minutes.

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Preferable inert gases utilized in the practice of the present invention are filtered compressed inert gas.
A preferable carrier gas useful in the practice present invention is one gas selected from the group of gases S consisting of car~on dioxide, room air, hydrogen, helium, and nitrogen. However, $t should be noted, that any compres ed gas substantially nontoxic to living materials may be utilized in a practice o~ the present invention.
The source of the carrier gas 6 is a source of gas capable Or generating a gas pressure of 30 pounds per square inch at the inlet 3 of the nebulizer 7. Preferred sources of compressed gas are compressed gAs cylinder, and laboratory electrolytic cell gas generators.

The nebulizer S utilized in the practice of the present invention may be any nebulizer 5 whi~h produces aerosols having droplet sizes o~ from 0.1 to about 3 microns in diameter. Although ultrasonic nebulizers, such as the LXB Instruments, model 108 may be used in the practice of the present invention, down draft or respiratory inhalat~on nebulizers of the Lovelace design are most preferred. The most preferred nebulizers o~ the present invention is a nebulizer used in inhalation therapy obtained frcm Inhalation Plastic. Inc., model 2S 4207, Or the Lovelace design. These nebulizers are single u~e, disposablQ units that generate aerosol droplets with median ma58 diameters in the range o~ two microns. ~hey require only a ~ew millimeters o~ solution to opera~&
e~iciently and generate dense mist having up to droplets per liter o~ gas.

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Wosl/0091s ' ' P~T/US9o/03663 Examplç~

Examples 1-3 are presented to demonstrate the optimal range of particle sizes effective in transforming target cells in the practice of the present invention.

Examples 4-8 are presented to de~onstrate the successful transformation of Chlamydo~onas reinh~L~
cells using the apparatus and methods o~ the present invention.

Exam~,e ,Aerosol Beam Microintection of Carboxy~luorescein into Chlamydomonas,reinhardtii usinc ? ~icro~ ~ss median diameter solute/solvent d~oplets ÇhlamYdomonas Fçinh~a~ii is a unicellular eucaryotic green alga. Cells of the wild type strain of Chlamvdomonas reinhardtii (ChlamydQmonas) were used as the target cells. The ce$1s were cultured in tris-acetate-phosphate ~TAP) l~quid medium. (Harris, E.H.
Chlamvdomonas Source, ~o~k~ Academic Press, 1989). The cells were concentrated by centrifugation and resuspended in TAP at a concentration of 107 cells/ml. 100 microliters of the cell suspension were subseguently plated onto an agar medium slab containing T~P and 1.5% by welght agar for consistency. The slab also lncluded an inert layer of Miracloth tCalbiochem, Co.) whlch was washed and steam autoclaved. Miracloth was includ~d to facllitate tha handling of the ~gar slabs. The slabs were thereafter incubated at 25C for from 1 to about 4 hours.
Following the incubation period the slabs were chilled at 4C for about an hour.

` .

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, The schematic of the apparatus used in the instant example is shown in Fig. 1. The aerosol was produced by a respiratory therapy nebulizer, model 4207 obtained from Inhalation Plastic, Inc. The mass med~an diamet~r of the aerosol droplets produced by the nebulizer was 2 micrometers, according to manufacturer~s literature. The nebulizing solution used was a bu~fered aqueous solut~on containing O.OlM sodium phosphate Ph 7.0 and 10 mg/ml of carboxyfluorescein. Both chemicals were obtained from the Sigma Chemical Company. The carrying gas was compressed nitrogen having a flow rate of 4 liters/min to achieve 30 psi in the nebulizer. The positive pressure created by the carrier gas was neutralized by opening the output piping of the nebulizer to the outside atmosphere. The piping leading to the vacuum housing (tubing 11 in Fig. 1) was positioned in close proximity to the open outlet piping of the nebulizer. The negative pressure in the system was created by a high-volume vacuum pu~p attached to the vacuum chamber. The vacuum pump used was a Marvac model R-10, set at 170 liters/min. The aerosol was accelerated into the vacuum housing through a nozzle having a diameter of 200 microns. The agar slab on which the cells were plated was placed on the target support platform, which, in turn, was positioned 1.5cm from the nozzle. The target support platform was rotated at 40 rpm, and advanced`at a rate of 333 micrometers~revolution.
The entire slab wa~ scanned timpacted by carrier gas or droplets) in about 3 to 4 minutes. A control group of cells ~non-impacted cells) was created on the slab by interrupting the aerosol flow in a regular pattern. Thus, the control cells on the slab are impacted with only carrier gas.

To determine the morphclogy of the cells impacted, the scanned slab was examined using a 40X dissection microscope. To determine the pattern of WO91/0091~ rcT/us9o/o3~3 carboxyfluorescein impact, the slab was examined using a W Products model T-33 longwave W transilluminator. To determine cell survival after impact, the slab was placed on a petri dish containing TAP ~edium and incubated for 2 days. To determine if microin;ection of carboxyfluorescein had occurred in the impacted cells, the cells were removed from the slab, washed, resuspended in TAP liquid medium, and examined with a Nlkon Labophot epifluorescence microscope, using a W f~lter cu~e to observe carboxyfluorescein fluorescence.

Results Following scanning the slab with 2 micron droplets no intact cells were observed in the impact areas.
Fluorescence was observed unifor~ly in all the impact areas. There was no growth in the impact area following a two day incubation. ~hus, it was determined that cells impacted by droplets having a mass median diameter of 2 microns or greater would not service the procedure.

Aerosol 9eam Microin~ection of carboxyfluo~çsein into Chlamvdomonas reinhardtii ~si~g Q~L_~iç~on maæs median diameter solute/soIvent dro~lets The protocol set forth in Example 1 was followed in Example 2 with the following except~on. A drying tube was included in the apparatus. The drying tubo was located in close proximity to the nebulizer, as shown in Fiq. 2. The drying tube had an internal diametar o~ l/2 inch and was 60 cm long. It contained silica gel which surrounded a central channel formed by a cylinder of 20 mesh stainless steel screening. The drying tube of the instant example was prepared by the inventox; however, similar drying tubes are available from TSI, Inc. (model 3062 Diffusion !~ - ' ' - .

.

~ ~, Dryer~. The solvent was completely removed by the dryi.ng tube so that only a dry aerosol of the solutes remained to be accelerated and impact the cells. Based on the mean masses of the solutes it is estimated that the impacting S projectiles had a mass.median diameter of about 0.1 microns.

Results Following scanning the slab with 0.1 micron projectiles, visual exa~ination with the 40X dissection microscope revealed that all the cells in the impacted area were intact. Further, the distribution of carboxyfluorescein on the slab was uniform throughout the impact areas. Thus, demonstrating that the cells has ben lmpacted. T~ere was 100% cell sur~ival after a 2 day incubation. However, no fluorescence was observed in the washed and resuspended impacted cells. This suggested that no carboxyfluorescein was successfully injected into the cells.
Example 3 Ae~Qsol Beam,Micr~iniecti,,on of Carboxyluorescein n~o ChlamydQ~Qn~,s_~eian~rd~ii on sorbitol containina medlu~--usi~g-~L5LJL~uIl mass media~ eter sQlute/so ~ent_droplets Example 3 ~ollowed thc protocol set forth in Example 1 with the ~ollowing exceptions: the agar slab lncluded 0.5M sorbitol to help os~otically stabilize the impacted cells; and the celis were immediately washed and resuspended following microscopic and W inspection of the slab. Thus, the incubation step, which determined cell survival was postponed until it was determined if carboxyfluorescein had been successfully microinjected into the cells.

Wosl/0091~ PCTlUS90/03~3 Res~lts Following scanning the slab with 2 micron droplets the cells were inspected wi~h the 40X dissection microscope and substantially all the cell5 in the impact area appeared intact. Following washing and resu~pension, substantially all of the impacted cells contained florescent carboxyfluorescein. Thu~, carboxyfluorescein was successfully microinjected into the cells. ~owever, following subsequent replating of the impacted cells, no cell survival was below 0.1%.

Exa~El~ 4 Transform~tion of Chlamydomon~s rein~r~S~ g53nt D15 throuah the mi~roiniection of D~A pl~smid p71.

The target cells were wild type Chlamy~omonas reinhardtii Mutant D15 which are incapable of growing on minimal media because of a deletion of the chloroplast tscA gene that renders them defective in photosynthesis.
No spontaneous reversion of this deletion has be~n observed. The DNA plasmid p71 used as the ge~etic transforming agent carries the ~hlam~domonas F-e.-i~h~L~
chloroplast DNA fragment Ecol8 (Harris, E.~. Chlamy~mQ~as Sou~e_~o~i~, Academic Press, 1989) cloned in E. ÇQ~ in thQ pUC8 cloning vector. It was obtained from Dr. Jans Aldrich, BP America. This fragment has the intact tscA
gene that wa~ deleted from the D15 mutant chloroplast DNA.

~he nebulizing solution included lOmM tris-Cl p~ 8.0, lmM disodium EDTA, 3mM magnesium chloride, O.lmg/ml plasmid p71, 10% polyethylene glycol 6000, and distilled water. All reagents were obtained from the Sigma Chemical Company. Polyethylene glycol and magnesium chloride are included in the solvent mixture to cause the tight condensation of the DNA, which protects it from shear .

2062~78 deqradation during aerosol formation. The effectiveness o~ this treatment was ver~fied in preliminary experiments in which the aerosol generated from this solvent was recovered and the DNA analyzed by electrophoresis for intactness. In addition, it was tested for biological function by transformation of ~. ÇQli-The target cells were grown in TAP liquid medium andccncentrated by centrifugation. The cells were plated on an agar slab and incubated as described in Example 1.
Before scanning, the ag~r slab was placed onto a petri plate containing solidified TAP medium containing 0.5M
sorbitol ~or two hours at room temperature and for an additional one hour at 4 C. It ls believed that the presence of the sorbitol in the medium helps the cells to maintain their integrity during an osmotically sensitive per~od during recovery after being impacted by the pro;ectiles.

The apparatus used in Example 4 is illustrated schematically in Fig. 2. The drying tube utilized was the same drying tube as used in Example 2. The laminar flow accounted for 50% of the total flow through the nozzle.
The nozzle was 200 microns in dia~eter. The target cells were positioned 1.5cm from the nozzle and the procedure used for scanning the surface o~ the agar slab was the same as was used in Examples 1-3. The c~rrier gas was nitrogen and the flow rate through the drying tube was set at 160 ml/minute.

Following the scanning of the surface of the agar slab, the impacted cells were screened for the presence of transformants. The slab was transferred to tris-minimal medium (Harris, E.H., Chlamydomonas Source 900k, Academic Press, 1989) and incubated in light. Thus, only those ce~ 1B which had been trans~ormed could photosynthesize in the presence of light and survive. Following incubation W~91/0~15 PcT/usso/o3663 for two days, two colonies were observed on the minimal medium. These colonies were removed and streaked onto petri dishes containing tris-minimal medium.
Subsequently, a plurality of colonie5 grew. This growth test was repeated a second time after seven days.

Total cell DNA was isolated from ~he trans~ormed ChlamYdomonas reinhardtii cells. The cells were grown to a concentration of 3 X lO cell~lml in TAP liquid medium.
They were harvested by centrifugation at 5000 Xg for five minutes and resuspended to a final concentration of lO
cells per ml in ic~ water buffer I (lOmM sodium chlor~de, lOmM tris-Cl, lOmM sodium EDTA, pH 8.0). This suspension was incubated with an equal volume of ice cold 4H lithium lS chloride and incubated for thirty minutes. The cells were then harvested by centrifuqation as before and then washed twice with ice cold buffer I. The cell pellet obtained in the final wash was weighed and then the cells resuspended in lO ml per gram pellet of ice cold buffer ~. One third volume of lO~- (w-v1 sodium dodecyl sulfate detergent was added along with O.l mg/ml of Pronase ~Sigma). This mixture was incubated at 37 C for three hours or longer until the chlorophyll was entirely converted to pheophytin (olive-green in color). This solution was then cooled to room temperature and extracted twice with freshly distilled phenol (the phenol was distilled, washed with one half volume of O.5M tris base and equilibrated before use with one volume of lOmM tris-Cl pH 8.0, lmM disodium EDTA, 50mM sodium chloride ). The aqueou~ phase ~eparated ~rom the second phenol extraction was mixed with one half volume of 7.5M ammonium acetate and then centrifuged in a micro centrifugQ (Eppendorf) for one minute. The supernatant was thereafter ~ixed with two ~olumes of ethanol and placed in -70 C freezer for ten minutes. The precipitated nucleic acids were collected by centrifugation in the micro centrifuge for o~e minute and ... . .

the supernatant discarded. The pellet was resuspended in one tenth of TE buffer (lOmM tris~Cl, l~M disodium EDTA, pH 8.0). This ammonium acetate precipitation step was repeated once more.

~ he DNA obtained from the transformants was thereafter analyzed. One aliquot of the DNA obtained as set forth above from one of transformants was digested with restriction enzyme EcoRI, one with a combination of Ba~HI and aglII and one with SmaI, using the enzymes according the manufacturer's instructions t~oehringer-Mannheim). A similar digestion was performed on DNA from wild-type cells, DNA from the D15 mutant cells, and DNA
from the p71 plasmid used in the trans~ormation experiment. The DNA fragments were separated by electrophoresis in 0.7% agarose gels using tris-borate-EDTA buffer according to standard procedures. The DNA was transferred to Nytran membranes (Schleicher and Schuell) using the standard Southern blot procedure. DNA for use as probes to detect specific fragments on the membrane was radioactively labeled by random priming, using a kit according to the manufacturer's instructions (~oehringer-Mannheim). Probes were prepared from pUC18 plasmid DNA
and from fragments of the Eco-la chloroplast DNA isolated from the p71 p}asmid.

The hybridization with the pUC probe roveals the pr-sence of an intact pUC vector sequence in transrormants. No reactivity was seen in ~he wild type or D15 strain DNA samples. These results demonstrate that the DNA was microinjected into the target mutant cells to produce transformants in the experiment.

Whlle the invention is susceptible to various modifications and alternative forms, specific embodiments thereo have been shown by way of example in the drawings w~9l/oo9ls PCT/US90/03663 -25- 20~2178 and have been described in detail. It should be understood, however, that it ig not intended to limit the invention to the particular forms disclo5ed, but on the contrary, the intention is to cover all modifications, eguivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims.

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Claims (13)

1. A method of introducing exogenous material into a living cell, the method including the steps of:

a) producing aerosol particles comprising said exogenous material;

b) accelerating said particles to a speed enabling them to penetrate and enter into said cell upon impact therewith; and c) impacting said living cell with said accelerated aerosol particles.

2. The method of claim 1 wherein said exogenous material is DNA.

3. The method of claim 1 wherein said aerosol particles include a solvent.

4. The method of claim 3 wherein the solvent is alchol based.

5. The method of claim 3 wherein said solvent is combined with colloidal gold, said colloidal gold being included to adjust the density of the resulting aerosol particles.

6. The method of claim 3 wherein at least a portion of the solvent is removed from the aerosol to dry the particles of exogenous material.

7. The method of claim 3 wherein said aerosol particles have a diameter of from about 0.1 to about 2 microns.

8. The method of claim 1 wherein said aerosol particles are accelerated by passing said aerosol particles from an area of higher gas pressure through an aperture having a diameter of from about 100 to about 300 microns and into an area of lower gas pressure.

9. The method of claim 8 wherein the gas pressure in said high gas pressure area is about 1 atmosphere and the gas pressure in said low gas pressure area is about 0.01 atmospheres.

10. The method of claim 1 wherein said speed is from about 400 to about 2000 meters/second.

11. The method of claim 1 wherein said living cell is a plant cell.

12. The method of claim 11 which further comprises the step of culturing said impacted living plant cell to grow into a plant.

13. The method of claim 12 which further comprises the step of screening among the progeny of said impacted living plant cell for transformed progeny.