Gene Transfer by Biolistic Process

Priscilla A. Furth

Abstract

injection and needle and syringe injection can be used to deliver both viral and nonviral vectors. Both jet injec-

injection has been most widely used in muscle tissue. The choice of which biolistic process to use is depen- dent on the specific application.

Index Entries: Biolistic process; jet injection; micropa~icle bombardment; needle and syringe injection; gene transfer; gene therapy; genetic immunization.

1. Biolistic Processes for In Vivo and In Vitro Gene Transfer

Biolistic processes have been developed for both in vivo and in vitro gene transfer. In this article, the term "biolistic process" is defined as the use of a physical force to transfer genetic material into a cell. Two types of force have been used: jet injection and microparticle bombard- ment. A third technique that does not require force, needle and syringe injection, has also been used to transfer genes into somatic tissues.

This article will initially focus on the use of jet injection for gene transfer. Then, because the three techniques listed above are used for similar applications, the advantages and disadvantages of each technique will be discussed.

2. Somatic Cell Gene Transfer by Jet Injection

Jet injection is the use of a pressurized column of fluid (jet) to accelerate genetic material into differentiated tissue for cellular transfection (1). In this technique, the jet itself is the force that

enables the genetic material to enter the cell. The efficiency of transfer by jet injection is correlated with the force of injection (2). The cells that express the transfected genes lie in a narrow path around the path of the jet injection. Expressing cells can be found up to 2 cm distant from the site of injection. For example, the jet injection can be applied at the skin surface to transfect underlying fat or muscle cells. The length of the jet injection path is dependent on the volume, the initial force of the jet, and the tissue that is injected. In passing through tissue, the jet loses force secondary to tis- sue resistance. When the force is dissipated, the solution pools. Few transfected cells are found in the vicinity of this pooled material.

Gene transfer by jet injection can be performed in both living animals and in explanted tissue cul- tures (1-4). It has been successfully used for gene transfer into mice, pigs, chickens, sheep, and rabbits. Minimal tissue damage is incurred.

A variety of solutions and genetic vectors can be used in conjunction with jet injection (5). The genetic vectors that have been used include naked

Address to which all correspondence and reprint requests should be addressed. Division of Infectious Diseases, Department of Medicine, University of Maryland Medical School, the Baltimore Veterans Affairs Medical Center and the Institute of Human Virology, 725 West Lombard Street, Room 545, Baltimore, MD 21201 USA, e-mail: furth@nih.gov

Molecular Biotechnology �9 Humana Press Inc. All rights of any nature whatsoever reserved. 1073-6085/1997/7:2/139-143/$9.25

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plasmid DNA, plasmid DNA incorporated into a complex with specific viral or cellular proteins (6), and viral vectors, such as adenovirus. Differ- ent vectors may be combined in a single solution for cotransfection experiments. The solution employed for jet injection is chosen for compat- ibility with the vector to be used, and can include a marker dye, such as india ink (3) or "DiI" (2) to visualize the path of injection.

The prototype jet injector is the Ped-0-jet (Stirn Industries, Dayton, NJ). This injector was devel- oped in the 1960s for the delivery of protein vac- cines into humans. The same injector is now used to deliver genetic material into various animal tis- sues. Other manufactured jet injectors can be used successfully, although it is essential that they deliver the jet with sufficient force. If the force is inadequate, there is poor gene transfer and the possibility of "blunt force" tissue injury. If the force is too great, aerosol is generated, which will dissipate in the environment and have no tissue penetration.

A number of different tissues have been suc- cessfully transfected using jet injection. The list includes skin, muscle, liver, and mammary gland. Theoretically the technique can be applied to any tissue that can tolerate the force of the jet. When adapting the technique to a new tissue, it is neces- sary to determine the optimal transfection force and volume based on connective tissue content. For example, liver, mammary gland and muscle require different forces. Liver requires the least force and muscle the most. Too much force applied to the liver can shear the tissue. Too little force applied to muscle results in blunt tissue injury.

2.1. Applications for Gene Transfer by Jet Injection

Transient transfection of plasmid DNA by jet injection can be used for several research applica- tions. First, optimal parameters for jet injection of a selected tissue are established by utilizing reporter genes such as chloramphenicol acetyl- transferase (CAT), [3-galactosidase, or luciferase (5). Then the technique can be used to evaluate tissue-specific or differentiation-specific param- eters, which cannot be approached in tissue-

culture cells. For example, expression of some classes of genes, such as milk protein genes, is dependent on the extracellular matrix or develop- mental state of a cell (7,8). Reporter genes linked to milk protein gene promoters can be used to evaluate tissue-specific promoter activity or to map promoter elements in differentiated mam- mary epithelial cells after jet injection (1). RNA and protein expression can be examined (3). Such an approach could be used to study tissue-specific alternative splicing or protein processing. For example, transfection of mammary epithelial cells in a lactating sheep results in secretion of the expressed protein in the milk (3). Finally, candi- date DNA constructs for generation of transgenic animals can be tested before injection into fertil- ized oocytes.

Genetic immunization is a practical use for jet- injected transient transfection. It is a rapid and efficient method to induce protein expression in both skin and muscle for the generation of anti- bodies (3). It is an inexpensive strategy for immu- nization of livestock. Plasmid DNA is stable, relatively inexpensive, and can be rapidly deliv- ered using one of the available jet injectors.

Transient transfection by jet injection can be used to deliver and test gene therapy strategies. It is particularly cost-effective for compar- ative studies of different genetic constructs in ani- mal models.

Stable transfection of plasmid DNA can also be accomplished through jet injection. The cot- tontail rabbit papillomavirus (CRPV) genome can be jet-injected into the skin of rabbits where it is stably expressed for months, and results in forma- tion of warts and tumors (4,9). This approach works with both CRPV genomic DNA isolated from a plasmid vector and religated, and when the entire plasmid vector including both bacterial and CRPV sequences is injected and has been used to map functions of selected papillomavirus gene products (10--13). For this approach to work for the expression of nonpapillomavirus gene prod- ucts, the vector may need to possess genes that offer transfected cells a proliferation advantage as well as genes that can direct intracellular vector replication (13).

It is possible to improve transfection efficiency of plasmid DNA by complexing the plasmid with cellular or viral proteins (6,14,15). Such an approach can be combined with jet injection to increase expression levels.

Jet injection can be combined with viral vec- tors for improved transfection efficiency or longer duration of expression. For example, DNA from a retroviral vector can be jet-injected to promote integration of the construct. Jet injection of aden- oviral vectors is an efficient and well tolerated means of delivering these vectors into tissues, al- though in this case gene delivery is most depen- dent on the adenoviral proteins and is relatively independent from the injection force.

2.2. Advantages and Disadvantages of the let-Injection Technique

The major advantages of the technique are its simplicity, cost, and adaptability. Aside from the injector, no special equipment is required. Plas- mid DNA is used, which can be rapidly prepared at a low cost. The type of solution is not critical. Manufactured injectors are available and are priced in the low thousands of dollars. These devices are very portable. The technique can be applied to a variety of tissues and animals.

At this time, the jet-injection technique is espe- cially useful for three areas of investigation: ge- netic studies in which there is no tissue-culture alternative, genetic immunization, and research applications that utilize sensitive techniques to evaluate gene expression. The major disadvantage of the technique is that transfected cells lie along a narrow path. Multiple injections must be used to transfect large areas of tissue, or the narrow path of transfected cells must be isolated from sur- rounding tissue for gene expression studies.

2.3. Future Developments Improving the efficiency of gene transfer by jet

injection is the most important goal. To this end, new models of injectors are being tested, which will provide rapid and repetitive injection of large areas of tissue. Development of replicating plas- mid based vectors would improve gene expres- sion levels derived from a single injection.

3. Somatic Cell Gene Transfer by Microparticle Bombardment

For microparticle bombardment, plasmid DNA is complexed onto particles, usually composed of gold or tungsten. The complexed particles are accelerated into cells using one of several accel- eration systems (16-18). In contrast to jet injec- tion, penetration through multiple cell layers is generally limited using this technique. Trans- fected cells usually lie within 500 gm of the injection site. For example, particles delivered at the skin surface do not generally travel beyond the epidermal layer (17).

Similar to jet injection, the technique can be used to transfect genes into somatic cells of both living animals and in tissue explant cultures (19,20).

Specialized techniques are used to prepare the DNA-coated particles. A few different accelera- tion systems are marketed (16-18). The experi- mental costs are generally higher than those associated with jet injection.

The technique has been successfully applied to a variety of different tissues and cells, including brain (20), skin, mammary gland, and liver (19) as well as tumor cells (21).

3.1. Applications for Gene Transfer by Microparticle Bombardment

Applications for gene transfer by particle bom- bardment into mammalian cells are basically the same as those described for jet injection. In addition to being utilized for gene transfer into mammalian cells, particle bombardment has been extensively used for delivering genes into plants (17).

Similar to jet injection, particle bombardment has proven to be a useful method for genetic immunization (18,22-26).

3.2. Advantages and Disadvantages of Microparticle Bombardment Technology

The major advantages of the technique is its adaptability to a wide range of tissues. Major dis- advantages are the poor tissue penetration, the efforts required for microparticle preparation, and the cost of acceleration systems.

3.3. Future Developments for Microparticle Bombardment

Manufacturers are actively working on devel- oping lower cost portable acceleration systems in addition to improved methods for microparticle preparation. Improving tissue penetration is an active area of research. Similar to jet injection, the technique can be combined with replicating viral vectors or viral vectors that can integrate for higher levels or longer durations of gene expression.

4. Somatic Cell Gene Transfer by Needle and Syringe Injection

The use of a needle and syringe for plasmid DNA gene transfer into somatic tissues of living animals is both simple and inexpensive. Reported transfection efficiency of either skeletal or cardiac muscle is comparable to either jet injection or microparticle bombardment (27,28). Successful transfection efficiency into liver (29) and thyroid gland (30) has also been reported. Transfection into skin (31) can be performed, but appears to be less reliable than either particle bombardment (22) or jet injection (1). In mammary gland, gene transfer by jet injection is clearly more efficient than needle and syringe injection (1,3). The effi- ciency of plasmid DNA transfer into muscle by needle and syringe injection can be affected by the type of solution used (27,32).

4.1. Applications for Gene Transfer by Needle and Syringe Injection

The two major applications for needle and syringe injection have focused on the relatively good efficiency of the technique in muscle. This approach has been used to map promoter elements in muscle tissues (33) and to effect genetic immu- nization after muscle injection (34-36). Genetic immunization has also been reported after needle and syringe injection into skin (31).

4. 1.1. Advantages and Disadvantages of Needle and Syringe Injection for Gene Transfer

The obvious advantage of this technique is its low cost and accessibility. For muscle tissues, it is an excellent option. The major disadvantage is its relative inefficiency in nonmuscle tissues.

5. Summary Two major biolistic processes are available for

use in studies utilizing gene transfer into somatic cells: jet injection and microparticle bombard- ment. Both methods can be used to map genetic elements, evaluate tissue-specific gene expression, deliver gene therapy, and effect genetic immuni- zation. Jet injection is relatively simpler and less expensive to perform than microparticle bombard- ment and exhibits better tissue penetration. Direct needle and syringe injection into muscle is a third, and less costly option for genetic immunization experiments or other investigations in muscle tissue.

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