Gene gun technology

Related Terms

Air pistol, biolistic particle delivery, biolistics, biological ballistics, DNA immunization, DNA plasmids, DNA vaccination, gene therapy, gold, hand-held gene gun, HeliosT gene gun, helium, immunotherapy, microprojectile method, PMGT, particle bombardment, particle-mediated gene transfer, transfection, transgene, titanium, tungsten, vaccine production technique.

Background

Genes are considered the building blocks of life because they provide instructions for all the cells in the body. Genes, which are located inside cells, control an organism's development and functions by instructing cells to make new molecules (usually proteins). Genes are passed down from parents to their children.
The gene gun technique, also known as biolistic particle delivery, biolistics, or particle bombardment, was developed in the early 1980s. This technique has revolutionized the science of genetic engineering, which involves manipulating an organism's genes. Gene gun technology involves delivering DNA or RNA coated on microscopic gold or titanium particles (bullets) into living tissues. The high-velocity acceleration of particles using the gene gun may be provided by compressed gas, centripetal force (external force to move a body along a curved path), electric discharge, or firing explosives. Acceleration provides the necessary force to puncture the cell membrane and deliver the materials into the cells of living tissues.
Gene guns are used to inject or deliver genetic information into the tissues of plants and animals to physically transform the target tissue. Transformation is the genetic alteration of a cell due to the incorporation or uptake of foreign genetic material. This alters the genetic makeup of the injected organism. For example, characteristics such as drought resistance or pest resistance can be incorporated into plants.
Gene gun technology can transform the tissues into stable and unstable forms. In stable transformation, the foreign genetic material is distributed equally to daughter cells, which are the cells that result from cell division. In unstable transformation, however, the foreign genetic material is distributed only to one of the daughter cells with each cell division. After 7-8 divisions, the foreign genetic material is not exhibited, hence, the expression of the new gene is transient (short term).
Gene gun technology also helps in gene therapy and DNA vaccination, which is also called genetic immunization. Gene therapy, an experimental procedure, involves replacing defective genes that may cause disease with normally functioning genes to treat or prevent illness. DNA vaccination is a method of protecting an organism from a disease by using genetically altered DNA to produce an immune response.

Methods

General: Gene gun technology is used to deliver genetic information into the tissues of plants and animals to alter the genetic makeup of the target tissue. This facilitates the incorporation of beneficial features such as drought resistance or pest resistance in plants, for example, or may be used for the purposes of gene therapy. Gene therapy, an experimental procedure, involves replacing defective genes that may cause disease with normally functioning genes to treat or prevent illness.
With gene gun technology, heavy microscopic particles such as tungsten, gold, or titanium are coated with DNA or RNA. The particles are accelerated into a plant or animal tissue at a very high velocity (1,400 feet per second or 300-400 meters per second) because the cell membrane has to be punctured. The accelerated particles penetrate the cells into the cytoplasm or the nucleus. The gene gun apparatus has a plate that allows only the DNA-coated particles to accelerate towards the target tissue, facilitating accurate transformation. Transformation is any alteration in the genome of a cell after the uptake, incorporation, or expression of foreign genetic material (DNA). Gene gun technology is conducted using two main methods.
Microprojectile gun method: The microprojectile gun method involves the use of a gas-driven gene gun that launches high velocity microprojectiles or microparticles, which are small particles that are composed of metals such as tungsten or gold. The microparticles carry the foreign genetic material into living cells. The DNA is first coated on microparticles and then delivered into the tissues using a gene gun. The size of the beads ranges from 0.5-5 micrometers. The metal particles penetrate several layers deep into the target tissue, depending on their velocity and momentum.
HeliosT gene gun: The HeliosT gene gun, developed by Bio-Rad Laboratories, Inc., is a hand-held device that is convenient to use because it allows rapid transfer of genes into various targets in a living organism (in vivo). It uses a low-pressure helium pulse (100-600 pounds per square inch) to deliver DNA- or RNA-coated microscopic particles into the target tissue. The gene gun does not require vacuum, which allows larger tissues, such as a human arm, to be transformed. The other advantages include the long shelf life of the cartridge and a shorter procedure time. The process is completed in less than five seconds, making it a useful approach for multiple gene deliveries. One major advantage of this technique is that it can coat various nucleic acids onto the gold; therefore, it can deliver different traits in a single experiment. Nucleic acids are long strands of nucleotides and a large number of nucleotides form DNA.
The HeliosT gene gun is particularly useful for research efforts to better understand and develop treatments related to gene therapy, as in cancer biology, wound healing, and several diseases.

Research

General: Several studies are being conducted on humans to evaluate the gene gun. The gene gun faces the challenge of manufacturing complexities but has shown promise in clinical studies. Gene gun technology has the potential to be used for a wide spectrum of clinical applications, both in vitro (a test performed in a test tube in laboratory) and in vivo (within the living organism). However, currently it is being used mainly for DNA vaccination and gene therapy. Gene therapy, an experimental procedure, involves replacing genes that can cause disease with normally functioning genes to treat or prevent illness.
Gene therapy: Gene gun technology is being studied in the treatment of several disorders such as bleeding disorders. For example, genes that produce clotting factors can be introduced into individuals with bleeding disorders, or genes that increase the production of red blood cells could be introduced into individuals with anemia. Also, several studies are being conducted to evaluate the use of gene gun technology to treat different types of cancer, liver, and heart diseases.
DNA vaccination: The gene gun technique was first developed for plant tissues but was later used on animals for DNA vaccination (also called genetic immunization). DNA vaccination is a method of protecting an organism against a disease by using genetically altered DNA to produce an immune response. Vaccines are preparations used to improve an organism's defense (immunity) against a particular disease. Genetic immunization is an alternative to antibody production. For example, a vaccine was delivered using a gene gun on the skin of mice for generating protective antibodies instead of injecting it into the muscles or veins.
Gene gun technology enhances DNA vaccination because it requires only a small amount of DNA to deliver the vaccine. Administration of DNA vaccines via gene gun has emerged as an important form of antigen-specific immunotherapy that involves the use of immune-strengthening techniques to fight illness. DNA vaccination using gene gun technology is being used for various applications.
Researchers are using gene gun-mediated transformation of cabbage to study the potential of the technique to provide insect resistance to vegetables and other plants. This could help to avoid the use of pesticides or insecticides on plants. Long-term exposure to pesticides may cause cancers, birth defects, and nervous system problems. Gene gun technology may help prevent defects related to pesticide exposure because it may provide insect resistance to plants, thereby reducing or eliminating the need or use of pesticides.
Gene gun technology has been used to deliver recombinant adeno-associated virus as a tumor vaccine in individuals with lymphoma. Lymphoma refers to various, usually malignant, tumors that arise in the lymph nodes or in other lymphoid tissue. The vaccine delivered using the gene gun technology enhances humoral responses in the injected individual. This facilitates the injected individual to fight against the tumors and thus prevents the complications related to tumors. Humoral immunity involves the secretion of antibodies to the antigens (foreign materials) by the B lymphocytes (blood cells).
Gene gun vaccination of stress protein heat shock protein (HSP) 70 is being studied to understand its role in increasing the depigmentation in vitiligo. Vitiligo is a chronic skin disease in which the loss of pigment in the skin leads to formation of irregular patches. This might help in developing treatment strategies against the skin disease.
Gene gun technology is also being used to study the efficacy of vaccines targeted against certain human viruses such as orthopoxvirus and the flu virus. Orthopoxvirus is a genus of poxviruses, which commonly affect mammals (e.g., smallpox). Researchers are conducting studies to immunize mice against the flu virus with pieces of DNA using gene gun technology.

Implications

General: Whereas many tissues are difficult to transfect using classical methods such as electroporation, using a gene gun is simple and efficient. Transfection is the process of introducing foreign material into the cells of animals or plants using a virus vector or other methods of transfer. Vectors are carriers into which foreign genetic materials (DNA/RNA) are inserted. The vector containing the foreign DNA/RNAs is then introduced into a recipient or target cell and maintained for study or expression. Gene gun technology has been developed as a nonviral method of gene transfer into various tissues. In the gene gun method, biologically inert particles such as metal atoms (tungsten or gold) coated with DNA or RNA serve to transfect the target tissue. Administration of DNA vaccines via gene gun has emerged as an important form of antigen-specific immunotherapy that involves the use of immune-strengthening techniques to fight illness.
Vaccines: DNA vaccination is a method of protecting an organism against a disease by using genetically altered DNA to produce an immunological response. This is a new and powerful strategy to generate immunological responses against infectious diseases and cancer.
Gene gun technology has been used as one of the methods for DNA vaccination, which induces humoral and cellular immune responses. Humoral immunity involves the secretion of antibodies to the antigens (foreign materials) by the B lymphocytes, which are a type of white blood cell. Cellular immunity does not involve the secretion of antibodies. Rather it destroys antigens by activating the T lymphocytes, another type of white blood cell, and macrophages, cells derived from white blood cells that engulf and digest the infectious agents. DNA vaccination occurs due to the presentation of antigens to the host's immune system in a natural form, similar to that achieved with live attenuated vaccines. Live attenuated vaccines are developed from the naturally occurring germs. The germs in the vaccines can still infect the people but cannot cause the disease because they are weakened (attenuated) in the laboratory (e.g., BCG vaccine against tuberculosis).
Plants: Gene gun technology may be used to genetically modify plants for various reasons. Many foods in the United States, including corn, soybeans, and canola, are genetically modified using various techniques including gene gun technology. The incorporation of iron and vitamins into rice, for example, may reduce the prevalence of malnutrition globally. Crops may also be genetically modified to withstand harsher temperatures than non-modified foods. As a result, these crops may survive in areas of the world where crops are difficult to grow.
Not all genetically modified plants are grown as food crops. Using gene gun technology, plants, including trees, have been genetically modified to help reduce groundwater pollution. These plants are designed to reduce the amount of heavy metal in contaminated soil, thereby reducing pollution.
In addition, crops such as rice, corn, soybeans, sweet potatoes, apples, tomatoes, cantaloupes, and other fruits and vegetables, have been genetically modified using gene gun technology to improve taste, color, size, and overall quality; reduce maturation time; increase nutritional value; increase tolerance to extreme temperatures; and improve resistance to diseases, pests, and herbicides.
Therapeutic applications: Gene gun technology can be used to introduce genes that will provide instructions for making proteins that might be therapeutic. For example, genes that produce clotting factors can be introduced into individuals with bleeding disorders, or genes that increase the production of red blood cells could be introduced into individuals with anemia.
Others: In addition to crops, animals have also been genetically engineered. For instance, researchers can genetically alter animals, including chickens and cows to increase their productivity of meat, eggs, and milk. They have also used it to improve the animals' health and feed efficiency.

Limitations

General: Gene gun technology has certain disadvantages that may be related to the method followed or to the adverse effects following gene transfer on plants or animals. One major drawback with the gene gun technology is the lack of sustained expression of the introduced genes in the target tissue. Therefore, multiple administrations are required in many cases.
The currently used gene gun methods may not accurately transfer genes to tissues of certain solid organs such as liver or brain because the system loses efficiency with deeper penetration. Therefore, modifications may be required in the gene gun methods to produce reliable results in such cases.
Allergy: Researchers speculate that introducing a gene into a plant using gene gun technology may create a new allergen, which may cause allergic reactions in sensitive individuals. Allergic reactions may also be triggered if modified foods contain genes from other organisms to which the consumer is allergic. For instance, researchers decided not to add a gene from the Brazil nut to soybeans because of the possibility that individuals allergic to Brazil nuts would have an allergic reaction to the modified soybeans.
Tissue death: A few studies have shown that delivering DNA directly into the tissues of the body using gene gun technology may cause death of the target tissue. This may be due to adverse reactions in the tissues to gold.
Ethical: Some people argue that artificially altering living things is a violation of the natural organisms' intrinsic value and that changing the genetic makeup of a living organism is ethically wrong. As gene gun technology gains popularity, new laws will be needed to address the ethical and social issues surrounding genetic alteration.

Future research

Heart disease: Researchers have conducted studies on directly injecting DNA plasmid into the heart. Although many complications have been reported, this technique has the potential to be effective for gene transfer into the heart and to be used as gene therapy for many heart diseases. Plasmid is a structure in the cell that consists of DNA and can exist and multiply independently of the chromosomes. Gene therapy, an experimental procedure, involves replacing defective genes (that could cause disease) with normally functioning genes to treat or prevent illness.
Liver: Scientists are studying gene gun technology to treat liver disorders as a potential alternative to hydrodynamics-based transfection. Hydrodynamics-based transfection is a simple method that involves the injection of DNA through the veins to transfer genes into an organism. Transfection is the process of introducing foreign material into animal or plant cells using a virus vector or other methods of transfer. Although certain complications such as death of the liver cells have been reported in some studies, the gene gun technology with few modifications has the potential to be used as gene therapy for liver diseases.
Cancer: Several studies are being conducted to use gene gun as a strategy to deliver DNA vaccines directly into the skin. This approach enhances the potency of the vaccine and might be especially useful in treating tumors. Synthetic polymer particles like poly methyl methacrylate (PMMA) have the potential to initiate immune responses by producing inflammatory cytokines, which are a type of protein used in cellular communication. Some studies have indicated that these particles have antitumor protective activity. Hence, scientists are evaluating gene gun technology for the delivery of PMMA particles mixed with plasmid DNA (DNA vaccination) to treat cervical cancer.
Diolistics: Diolistics is the technique of incorporating fluorescent dyes into cells using a gene gun. This helps in the identification of the structure and function of the cells in tissues that are deeply located. Scientists are using diolistics to study the nervous system (nerve cells and neuronal genes) to examine the structure and function of the nerve cells. This may help to identify genes that cause certain nervous system disorders, thereby improving understanding of the diseases and potentially leading to new therapies.

Author information

This information has been edited and peer-reviewed by contributors to the Natural Standard Research Collaboration (www.naturalstandard.com).

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