MicroprojectileBombardmentJulie Russell Kikkert, Cornell University, New York, USA

Microprojectile bombardment is a method by which foreign substances such as DNA are

introduced into living cells and tissues via high-velocity microprojectiles.

Introduction

Cell walls and membranes restrict the introduction ofmacromolecules into cells. Genetic engineering requiresthat nucleic acids pass these barriers and reach the nucleusor certain other cellular organelles. In the early 1980s,genetic engineering of plants was limited to a few species.Different approaches were tried to breach plant cell wallsand plasma membranes, such as enzymatic degradation ofthe cell wall to create protoplasts, followed by transientmembrane disruption. However, such methods were notsuited to many species. Frustrated by the limitations oftechniques available at that time, Dr John Sanford atCornellUniversity inGeneva,NY,USA, began to test newways to pierce cells.Microlasers were used to cut tiny holesin cells, but the cytoplasm tended to leak out of the cellsinstead of the DNA diffusing in. In collaboration withother colleagues at Cornell (E.D.Wolf, N.D.Allen andT.M. Klein) the idea of shooting microscopic metal particlesinto plant cells to create holes and also to carryDNA insidewas developed and tested. The feasibility of this conceptwas demonstrated by shooting tungsten particles intoonion epidermal cells (Sanford et al., 1987; Sanford, 1988).The process was termed ‘biolistics’ (short for biologicalballistics) by the inventors, but is known by a variety ofterms including microprojectile bombardment, particlebombardment, the gene gun method and the particle gunmethod. Microprojectile bombardment has become areliable technique for genetic engineering of plants and avariety of other organisms. The method also has medicaland veterinary applications.

Outline of Methods

Basic concept

The basic concept put forth by Sanford et al. (1987) statesthat

Particles of the appropriate size, accelerated to the appro-priate velocities should readily penetrate thin barriers such ascell walls and cell membranes, thereby entering into the cell’scytoplasm. In this way, particles carrying biological sub-stances could be introduced into cells.

Substances can be carried into cells by a variety ofmechanisms: (1) the substance can be coated onto thesurfaces of inert particles; (2) cells can be bathed in asolution and bombarded with uncoated particles; and (3)the substances can be contained in particle interiors(Sanford et al., 1987). Microprojectiles can be any minute,coherent particles capable of being accelerated such thatthey penetrate cells and tissues in a nonlethal manner.Most commonly, DNA is coated onto the surface ofmicrometre-size gold or tungsten particles by precipitationwith calcium chloride and spermidine. Once inside thecells, the DNA elutes off the particles and is expressed bythe host cells, and a portion is stably incorporated into thehost chromosomes.

Devices

Microprojectiles are accelerated and delivered into targetsby a device often referred to as a gene gun (Figure 1).Various gun designs have been proposed and tested(Sanford et al., 1987, 1991; Gray and Finer, 1993). Devicesthat employ a macroprojectile as a means of acceleratingparticles proved to be highly effective. The first commercialdevice, made available by Sanford and Wolf, employed abullet-like plastic cylinder as the macroprojectile. The

Article Contents

Secondary article

. Introduction

. Outline of Methods

. Applications

. Future Developments

. Summary

Figure 1 Demonstration of the use of a gene gun. The model shown is theprototype of the PDS-1000/He. Photo by Rob Way, Cornell University.

1ENCYCLOPEDIA OF LIFE SCIENCES © 2001, John Wiley & Sons, Ltd. www.els.net

microprojectiles, were placed on the face of the macro-projectile, which was then loaded into the barrel of thedevice. A blank .22 gunpowder charge was loaded behindthe macroprojectile. When the device was fired, themacroprojectile was accelerated at high velocity downthe barrel until it met a stopping plate with a small orifice.The stopping plate retained the macroprojectile, while themicroprojectiles were launched from its surface throughthe orifice of the stopping plate and towards the target.This design was later marketed widely by DuPont, Inc.(Wilmington, DE, USA) as the PDS-1000 device (PDSstands for particle delivery system).A second-generation device, the PDS-1000/He mar-

keted by Bio-Rad Laboratories (Hercules, CA, USA)employs a similar principle but uses compressed helium asthe driving mechanism (Figure 2). In this design, themacrocarrier is a film of high-strength plastic calledKapton and the stopping mechanism is a metal screen. Inthe PDS-1000 devices, the cells or tissues are placed in avacuum chamber to reduce deceleration of the particles byair friction.A third generation of deviceswere developed ashand-held units for tissues or organisms that would not fitinto or tolerate a vacuum chamber (e.g. animals). TheHelios Gene Gun System marketed by Bio-Rad Labora-tories employs an adjustable helium pulse to sweep DNA-orRNA-coated goldmicrocarriers from the inner wall of asmall plastic cartridge directly into the target. It is a hand-held, reusable unit that relies on an external source ofhelium. Powderject Vaccines, Inc. (Madison, WI, USA)has developed a small disposable, inexpensive unit drivenby a 5mL helium canister. It is designed for humanvaccination, but is not yet marketed commercially.

Applications

Because microprojectile bombardment is a physicalprocess and is not limited by the target cell type or species,

it has found broad application. Transgenic plants from avariety of species have beenobtainedwith thismethod. Forexample, transgenic papaya were developed with resis-tance to papaya ringspot virus and are being commerciallygrown in Hawaii, USA, where this deadly virus wasthreatening to destroy commercial production (Lius et al.,1997). Microprojectile bombardment has special applic-ability to monocotyledonous plants such as corn, rice andother cereals, which are often recalcitrant to differenttransformationmethods.Microprojectile bombardment isuniquely suited for transformation of organelles such aschloroplasts and mitochondria. Additionally, numerousfungal, bacterial, algal and animal species have beengenetically transformed with this technique. The methodhas been used to wound plant cells prior to infection withAgrobacterium tumefaciens (another DNA delivery meth-od), and as a method of viral inoculation. Microprojectilebombardment is being tested for somatic gene therapy andgenetic vaccination in humans and other mammals.

Future Developments

Many laboratories worldwide are working to improvemicroprojectile bombardment technology. We can expectimproved devices, and improvements in microprojectiledesign and coating methods. Procedures to enhance cellsurvival and stable transformation frequency are alsolikely to be developed. The technology will be extended tomore species, and delivery of substances other than nucleicacids.

Summary

Microprojectile bombardment is a reliable method forgenetic transformationof awide range of organisms.Othertechniques are also available and each investigator mustchoose the best method for a particular application.Microprojectile bombardment offers a number of desirablefeatures. It is broadly applicable; the methodology isrelatively simple and essentially the same regardless oftargets andDNAused; it is a physical process not involvingother organisms; it can be used for transient geneexpression studies as well as production of transgenicorganisms; and it can be used to transform cellularorganelles.

References

Gray DJ and Finer JJ (eds) (1993) Development and operation of five

particle guns for introduction of DNA into plant cells. Plant Cell

Tissue and Organ Culture 33: 219–257.

Lius S, Manshardt RM, Fitch MM-M, Slightom JL, Sanford JC and

Gonsalves D (1997) Pathogen-derived resistance provides papaya

Macroprojectile

Microprojectiles

Stopping screen

Compressedhelium

Figure 2 The microprojectile launch mechanism of the PDS-1000/Hedevice. A shock wave of helium gas launches the macroprojectile from itsholder. The macroprojectile slams into the stopping screen and is retained.The microprojectiles continue through the openings in the screen and hit atarget placed below.

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with effective protection against papaya ringspot virus. Molecular

Breeding 3: 161–168.

Sanford JC (1988) The biolistic process. Trends in Biotechnology 6: 299–

302.

Sanford JC, Klein TM, Wolf ED and Allen N (1987) Delivery of

substances into cells and tissues using aparticle bombardment process.

Particulate Science and Technology 5: 27–37.

Sanford JC, Devit MJ, Russell JA et al. (1991) An improved, helium-

driven biolistic device. Technique 3: 3–16.

Further Reading

Hagio T (1998) Optimizing the particle bombardment method for

efficient genetic transformation. Japan Agricultural Research Quar-

terly 32: 239–247.

Haynes JR, McCabe DE, Swain WF, Widera G and Fuller JT (1996)

Particle-mediated nucleic acid immunization. Journal of Biotechnol-

ogy 44: 37–42.

Johnston SA and Tang D (1994) Gene gun transfection of animal cells

and genetic immunization. Methods in Cell Biology 43: 353–365.

Southgate EM, Davey MR, Power JB and Marchant R (1995) Factors

affecting the genetic engineering of plants by microprojectile

bombardment. Biotechnology Advances 13: 631–651.

YangNS and Christou P (eds) (1994)Particle Bombardment Technology

for Gene Transfer. New York: Oxford University Press.

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