Heat-shock mediated transfection
Related Terms
Biolistics, DNA vaccination, electroporation, gene gun, gene therapy, gene transfer, genetically altered food, genetically modified organisms (GMO), heat-shock mediated transfection, laser transfection, lipofection, microinjection, nuclear injection, oligonucleotides, plasmid, transduction, transfection, transformation, viral vectors.
Background
Genes: Genes are made of deoxyribose nucleic acid (DNA) sequences, and are considered the building blocks of life because they provide instructions for all of the cells in the body. Genes, which are located inside the cells, control the development and function of an organism by instructing cells to make proteins and other nucleic acids. These gene products then perform all the tasks that make the cell function.
DNA is a double-stranded chain of nucleotides that is wound up in a spiral (helix). Thus, DNA is often called a "double helix." Nucleotides are the building blocks of DNA and are made of nitrogen bases, sugars (deoxyribose), and phosphate. Nitrogen bases are of two types in DNA: purines, such as adenine (A) and guanine (G), and pyrimidines, such as cytosine (C) and thymine (T). The sequence of bases in DNA contains the genetic code, or hereditary information.
While DNA contains the instructions for making proteins, these instructions must be first "transcribed" into ribonucleic acid (RNA) before they are made into proteins. RNA is a single-stranded nucleic acid that is read by the cell's protein-making machinery. The RNA is made of nitrogen bases, sugars (ribose), and phosphate. The nitrogen bases in RNA are made of purines, such as adenine (A) and guanine (G), and pyrimidines, such as cytosine (C) and uracil (U). RNAs that carry genetic instructions for proteins are known as "messenger" RNA or mRNA. There are many other types of RNA that are not made into proteins, but have regulatory functions themselves. These include ribosomal RNA (rRNA) and translational RNA (tRNA) that form components of the cell's protein-making machinery.
Gene transfer: Gene transfer refers to the transfer of genetic material from one organism to another. Organisms can inherit genes in two primary ways, the first being gene transfer from a parent to an offspring, which is called as vertical gene transfer. The second is gene transfer from one individual to another of the same or different species, which is called horizontal gene transfer. Horizontal gene transfer may happen naturally as an evolutionary process (such as drug resistance in bacteria) or may be done artificially.
When genes are transferred from one cell to another, they may integrate into the receiving cell's own genetic material. This alters the genetic makeup of the organism that received the new genes. For example, certain characteristics such as resistance to drought or resistance to pests can be incorporated into plants. The effect of gene transfer may be temporary (transient) or stable (permanent). In transient gene transfer, the transferred DNA does not get combined (integrated) into the genetic material within the nucleus (nuclear genome) of the recipient cell. However, high levels of the transferred gene (transgene) may be expressed. In stable gene transfer, the transferred DNA is integrated into the host genome, and the genetic information of the recipient cell is permanently changed.
Applications: Gene transfer has been found helpful in gene therapy, an experimental procedure that involves replacing defective genes that may cause disease with normally functioning genes to treat or prevent illness, and DNA vaccination (also called genetic immunization), a method of protecting an organism from a disease by using genetically altered DNA to produce an immune response. An immune response refers to body's resistance to an infection or disease-causing agent. For example, a DNA vaccine is used to produce an immunological response against infectious diseases and cancer.
Methods
Gene transfer: Transferring genes or foreign material (e.g., drugs) into cells may be done biochemically, physically, or by using a carrier or "vector" such as a virus. Transfection refers to the transfer of DNA or other agents by nonviral methods into animal cells. Transformation is the nonviral DNA transfer in bacteria and nonanimal cells (such as fungi, algae, and plants). Transduction refers to DNA transfer between bacteria through a virus carrier. Several methods of gene transfer are described below.
Liposomal method: A liposome is a small lipid (fat) vesicle that contains water or saline (salt solution) in the center. A vesicle is a small bag or pouch that is surrounded by its own membrane inside the cell. The layered walls of the liposomes are made of phospholipids (a type of lipid or fat) that are similar to the phospholipids found in the cell membrane. Introduction of DNA into cells via liposomes is known as lipofection.
During lipofection, nucleic acids enter the target cell by endocytosis, which is a process by which cells absorb molecules, such as liposomes, from the cell's outside environment by engulfing it with the cell membrane. In this process, the liposomes bond (adhere) to the target cell surface, followed by fusion of the liposomal membrane and target cell membrane at the bonding site (adhesion site). This interaction causes interruption at specific places (localized disruptions) of the cellular and liposomal membranes, which allow passage of the liposome contents into the cytoplasm of the cell, thus delivering the genes or biologically active materials into the target cell.
Different types of nucleic acids, such as DNA, RNA, oligonucleotides (short segments of DNA or RNA), and viral nucleic acids (viral DNA or RNA), may be delivered into a cell through lipofection. Additionally, nucleic acids of unlimited size can be delivered, ranging from single nucleotides to large mammalian artificial chromosomes. An artificial chromosome refers to an artificially constructed segment of a nucleic acid, which carries the genetic information for a particular organism. Liposomes may also be used to deliver drugs, vaccines, or enzymes (a type of protein) into the cells of an organism. Liposomes are generally considered nontoxic, biodegradable, and nonimmunogenic, meaning that they do not produce a specific immune response. Immune response refers to the body's resistance to an infection or disease-causing agent. Biodegradable refers to a substance that can be decomposed by natural means. For example, lipofection has been used successfully to deliver DNA into plant cells of several species, such as tobacco, petunia, and carrot, so as to introduce certain genetic characteristics in these plants that are beneficial, such as resistance to pests.
Chemical method: Several chemicals such as polyethylene glycol (PEG), polyvinyl alcohol, calcium phosphate, and diethylaminoethyloethyl-dextran (DEAE-dextran) have been used for direct DNA transfer into protoplasts, which are bacterial or plant cells that lack a rigid cell wall but still have an intact plasma membrane. In this process, the DNA to be transferred is mixed (suspended) in a solution with a high concentration of these chemicals and then mixed with protoplasts. The presence of these chemicals facilitates DNA binding to cell membranes and entry of the DNA into the cell via endocytosis.
The advantages of using chemical methods of DNA delivery include the simplicity of the process, ease of production, and relatively low toxicity, and the process does not require special equipment. However, there are few disadvantages associated with this method, including the high degree of rearrangements, which cause unusual forms of DNA that may lead to cell damage, and increased chances of multiple insertions of the same DNA in the target cells, causing damage to the cells. Calcium phosphate (a chemical) gene transfer is used more commonly for production of long-term stable transfection, whereas DEAE-dextran (a chemical) is used for transient (short-term) transfection. Chemical-mediated transfection has been used to incorporate and study gene expression in some plants, such as maize (corn) and rice. When cells are transfected by the chemical-mediated DNA delivery method, about 20% of them take up the foreign DNA. The rate of transfection (transfection efficiency) depends on several factors, such as the chemical used, the concentration of the DNA to be inserted in the chemical solution, the pH of the chemical solution, the size of the DNA to be inserted, and the type of cell into which the DNA is being inserted.
Electroporation: Electroporation is a method of introducing DNA into the cells by exposing the cells to high voltage electric pulses for very brief periods of time. In this process, the protoplasts derived from plants or bacteria are suspended in a suitable ionic solution (a medium that conducts electric current) that contains the DNA to be transferred. This mixture is exposed to electric pulses of a chosen voltage for the desired time, based factors such as the size of DNA to be inserted and target cell. The electric pulses temporarily disrupt the phospholipid bilayer of the cell membrane (making holes in the membrane) and allow the DNA molecules to pass into the cell.
Generally, low voltage and long pulses produce high rates of temporary (transient) transfection, while high voltage and short pulses give high rates of stable (long-term) transfection. The advantages of electroporation are its simplicity, its high efficiency, its effectiveness with nearly all cell and species types, the ability to insert DNA into multiple samples in one experiment, and that it may be performed with intact tissue, i.e., in the tissue of a live organism. However, some of the disadvantages include: cell damage if the pulses are applied for an extended period of time, if the pulses are the incorrect length, or if they are of the wrong intensity; cell damage if nonspecific transport of material into and out of the cell occurs during the time of the disruption of the cell membrane (making a hole in the cell membrane); and the fact that only small DNA sequences (5-20 kilobases or kb) may be transferred. Using the electroporation method, successful transfer of genes has been achieved with the protoplasts of tobacco, petunia, maize, rice, wheat, and sorghum so as to introduce certain genetic characteristics in these plants that are beneficial (e.g., resistance to pests).
Biolistics: Biolistics, also known as particle gun method or gene gun technique, is a process of introducing DNA or RNA into living tissues by coating microscopic gold or titanium particles (bullets) with the nucleic acid and using a gene gun to force the particles into the skin of the organism. The high velocity 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 the particles so that they can puncture the cell membrane, which allows the particles to enter into the cells of the living tissues.
The advantage with biolistics is its flexibility in reaching different target tissues, its effectiveness in nearly all cell and species types, and its ability to be used in a wide range of applications (e.g., gene therapy). The disadvantages of this method include the need for special equipment, a high degree of rearrangements (which cause unusual forms of DNA that may lead to cell damage), and a high frequency of multiple insertions of the same DNA. For example, the biolistics method of DNA delivery has been used to transfer the cry gene (which provides instructions for the production of cry (crystal) proteins from Bacillus thuringiensis that are responsible for the insecticidal (fatal to an insect) activities of bacterial strains) into maize for resistance to the European corn borer (a crop insect).
Microinjection: Microinjection is a gene transfer method, where the DNA solution is injected directly inside the cell or the cell nucleus through an inserted cannula (a very thin glass tube or capillary glass micropipette). The process is observed and manipulated under the microscope. This process is usually done in cells that lack a cell wall, including protoplasts, the cells of an embryo, meristems (the growing tips of plants), and germinating pollen (the sprouting of a seed or spore), since a cell wall can interfere with the injection of DNA using this technique.
The advantages of the microinjection DNA delivery method are its high flexibility to reach different structures or tissues, visual control of the manipulation through the use of the microscope, and its use in wide range of applications (e.g., gene therapy). However, the process is slow, requires specialized and expensive instruments, and requires highly skilled and experienced personnel. This method has been used to study gene expression in artificially grown cells (cultured cells), and in the production of transgenic animals (genetically altered animals) such as chickens and cows, to increase their production of meat, eggs, and milk. This method is also used in in vitro fertilization, which is a laboratory procedure where sperm is placed with an unfertilized egg (ovum) to achieve fertilization (formation of embryo).
Laser method: The laser gene transfer method involves the transient disruption of cell membrane by femtosecond (fs, or one quadrillionth of a second) laser pulses. A laser is a device that produces a very narrow, high beam of light generally used for cutting. Temporary pores are created by focusing a laser beam for some milliseconds on the membrane. Through this pore, the DNA or RNA may enter the cell. This method of DNA delivery into target cells is highly efficient and applicable to a wide variety of cells from plants and animals. However, it requires special and expensive instruments and highly skilled and experienced personnel. Laser-mediated DNA transfer methods have been successfully used in plant cells and embryonic cells, i.e., the earliest stage of development in an organism.
Fiber-mediated method: The fiber-mediated method of gene transfer involves DNA delivery into the cell cytoplasm (a cell component outside the nucleus) and nucleus (the central core of cell containing genetic material) by silicon carbide fibers, which are fibers usually 0.6 micrometers ((m) in diameter and 10-80 (m in length. In this process, the target cells and DNA to be transferred along with the silicon carbide fibers are mixed (suspended) in a culture medium (artificially grown cells in a medium). This mixture is rotated very fast (vortexed), during which the silicon fibers act as microinjection needles, puncturing the cell membrane temporarily and allowing for the delivery of DNA into the cytoplasm and nucleus of the cell. This method has been used successfully for gene transfer in maize and tobacco.
Heat shock method: The heat shock method of gene transfer involves intense short pulses of heat to the target cells, which temporarily disrupts the cell membrane, allowing the DNA to be transferred into the cell. This process is usually done along with the chemical-mediated method or electroporation for increased efficiency in DNA transfer. The advantages of this method are its simplicity and that it may be used with intact tissues. However, this method may damage the target cells due to very high temperatures. The heat shock method of DNA delivery has been utilized to study the expression of certain bacterial genes that allow for survival in high temperatures.
Vector-mediated method: Vector means a carrier; in the context of gene transfer, a vector is a circular DNA molecule, including plasmids, which are DNA or RNA molecules that are separate from the bacterial chromosomal DNA (genetic content of bacteria) and viral DNA. The plasmid DNA or RNA are capable of replication (multiplication) and existence independent of the host organism. A piece of foreign DNA (the DNA to be transferred, which is taken from another organism or which is synthesized) is inserted into the vector genome (genetic content), which can be carried and introduced into a recipient (target or host) cell. Plasmids, which are one type of vector, may be single- or double-stranded and circular or linear. Plasmids carry genes that provide a selective advantage on their host, such as resistance, i.e., the capacity of an organism to defend against a disease or an agent, such as certain toxins or antibiotics (anti-infective agents against certain infections). For example, the gene to be replicated or transferred is inserted into the plasmid vector, which contains genes that make cells resistant to particular antibiotics. Next, the plasmid vector transfers the gene to the target/recipient bacteria by conjugation, which is the process of transferring genetic material between bacteria through the temporary union of the cells. Then, the bacteria are exposed to particular antibiotics. Only the bacteria that take up copies of the plasmid survive because they contain the gene that makes them resistant to the antibiotic. Although a virus is used to transfer the DNA into a plant or animal, that organism is not at risk of developing an infection from that virus because the genes that control the ability of the virus to be infectious are removed.
Viruses that infect plants and animals have been used as vectors to efficiently transport the genetic material inside the cells they infect. Delivery of genes by viruses is known as transduction, and such infected cells may be described as transduced. Some of the viruses that have been used as vectors include retroviruses, adenoviruses adeno-associated viruses, and herpes simplex virus type 1. The Moloney murine leukemia virus (Mo-MLV), which has the ability to integrate into the host genome in a stable fashion and is capable of infecting both mouse cells and human cells, thus has been used in many clinical studies for gene therapy. Retroviruses are a group of viruses with genetic information in the form of RNA and have the enzyme reverse transcriptase; examples include the Moloney murine leukemia virus and the human immunodeficiency virus type 1. Adenoviruses are DNA viruses that cause conditions such as upper respiratory tract infections, eye infections (conjunctivitis), stomach and intestinal (gastrointestinal) infections, in humans; examples include the avian adenovirus and the bovine adenovirus. Adeno-associated viruses (e.g., dependovirus) are single-stranded DNA viruses and not known to cause any disease in humans. Herpes simplex virus type 1 (HSV 1) is a DNA virus that causes infection of the lips, mouth, and face in humans.
Certain properties of viral vectors make them advantageous over other methods of gene transfer, including the fact that they have a minimal effect on the natural biochemical reaction processes of the cell they infect, such as cell damage, thereby causing very low toxicity to the target cells, promoting the survival of the cells. Viral vectors may be modified in such a way as to minimize the disease-causing (pathogenic) features of the virus and also to target a specific kind of cell, thus increasing specificity. However, certain viruses, such as adenoviruses, can rapidly rearrange their gene structure and sequence (they are genetically unstable). The genetic instability of these viruses makes them unpredictable, increasing the possibility that they could interfere with the study of the gene that was inserted. Also, there may be a slight chance that the virus may recover its ability to cause disease in the infected organism, even if researchers think they have removed all of its disease-causing genes.
The genetic material, after being introduced into the nucleus or cytoplasm of a cell by various gene transfer methods described above becomes integrated (joined) with the host DNA; this is called transgene integration. Generally the transgene integration occurs at random sites on the host genome. There is also an increased chance of integration of multiple copies of the introduced gene sequence into the host DNA. Targeted gene transfer involves gene sequence integration at specific sites on the host DNA.
Research
Gene expression studies: Genes provide instructions for the making of proteins that are required for the growth and maintenance of the body. Genes are also responsible for certain characteristics that may be expressed physically and can be observed (e.g., hair color) or nonphysically and cannot be observed (e.g., resistance to toxins). Gene transfer has made a significant contribution to understanding the expression of genes that may be helpful in understanding disease processes. Thus, gene transfer techniques may help in prevention, treatment, and monitoring of diseases.
Gene therapy: Gene therapy is an experimental procedure that involves replacing genes that can cause disease with normally functioning genes so as to treat or prevent illness. Gene transfer techniques are being studied in the treatment of several diseases. For example, gene transfer may help treat diabetes (type 1), which is a chronic (long-term) disease involving excess sugar (glucose) in the blood due to insufficient production of insulin, a hormone from the pancreas. A hormone refers to a substance produced by certain tissues in the body and acting like a chemical messenger in the regulation and coordination of the activity of many organs in the body. Insulin regulates blood sugar levels in the body. The development of diabetes has been associated with both hereditary and environmental factors. The gene PDX-1 has been found to be important for insulin production. Using gene transfer technology, the PDX-1 gene has been transferred into mice, where the gene is activated or expressed by liver cells which produce insulin. The PDX-1 gene that was transferred may reprogram tissues other than the pancreas to make insulin and control the abnormally high blood sugar levels in patients with diabetes. Also, several studies are being conducted to study the use of gene transfer techniques, such as gene gun technology, to treat different types of cancer, liver, heart diseases, and bleeding disorders. Gene gun technology is a process of introducing DNA or ribonucleic acid (RNA) into living tissues by coating microscopic gold or titanium particles (bullets) with the nucleic acid and using a gene gun to force the particles into the skin of the organism.
Parkinson's disease: Gene therapy has been studied as a possible treatment for Parkinson's disease, a degenerative (progressively deteriorating) nervous system disease. Researchers have recently figured out a way to transfer genes into the brain using liposomes, which are small lipid (fat) vesicles (small bags or pouches that are surrounded by their own membrane inside a cell) that contain water or saline (salt solution) in the center. Liposomal transfer of DNA allows billions of copies of a gene to be inserted into the brain area called the subthalamic nucleus to calm its overactive cells. The genes transferred lead to the production of an enzyme (a type of protein) known as glutamic acid decarboxylase (GAD), which in turn speeds up (catalyses) a chemical reaction to produce a chemical substance called gamma aminobutyric acid (GABA). GABA acts as a direct inhibitor of the overactive cells of the brain. Researchers are hopeful that this method may be an effective treatment for Parkinson's disease in the future.
Retinal diseases: Studies are being conducted to address a disease of the retina (the light sensitive part of the inside of eye) that causes blindness from birth, by means of the delivery of a liposomal DNA complex to the retinal pigment epithelial (RPE) cells. RPE is a pigmented layer of cells near nerve (neural) cells of the retina that nourishes the retinal cells important for vision. The entry of the liposomal DNA into the RPE is generally not efficient. However, the delivery of liposomal DNA into the RPE is facilitated by the use of ultrasound (sound wave) energy.
Agriculture: Researchers are using gene transfer techniques in genetically altered cabbage to study the potential of the technique to provide resistance to insects in vegetables and other plants. If this is effective, the need for pesticides or insecticides would be reduced. Pesticides are chemical agents that kill the pests that cause disease and damage to the plants and animals. These pests include insects, bacteria, virus, fungi, and weeds. Insecticide is a type of pesticide that kills insects. This research is important since long-term exposure to pesticides may cause cancers, birth defects, and nervous system problems.
DNA vaccination: Genetic immunization, or 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. The DNA vaccine can be delivered using a gene transfer technique such as gene gun technology. During this process, the DNA being used to induce an immune response is coated onto gold or titanium particles, and using the force associated with gene gun transfer technology, the DNA is incorporated into the skin cells of the organism. This differs from more traditional methods of vaccination, which use a needle to introduce antibodies into a vein or the muscle of the organism. The DNA transfer technique (gene gun technology) enhances DNA vaccination because only a small amount of DNA is required to create an immune response. Administration of DNA vaccines via the gene gun technique has emerged as an important form of antigen-specific immunotherapy, i.e., the use of techniques that improve the body's immune system so as to fight illness, some of which techniques are described below. An antigen is any substance, such as a virus, bacterium, toxin, or foreign protein, which triggers an immune system response in the body. The immune response is in the form of proteins that are specific to an antigen, and these proteins are known as antibodies or immune bodies. DNA vaccination against several diseases, such as hepatitis B viral infection (liver disease) and human immunodeficiency virus (HIV), is showing positive results in animal studies, and further research is continuing in this area.
Lymphoma: Gene transfer techniques have been used to deliver recombinant adeno-associated virus as a vaccine against tumors (cancerous growths) in individuals with lymphoma. Lymphoma refers to various, usually malignant, tumors that arise in the lymph nodes or in other lymphoid tissues of the body's immune system, such as tonsils, spleen, and bone marrow. Lymph nodes are small, round structures in the body that act as a defense mechanism against harmful microorganisms and toxins that may enter the body. Lymph nodes perform this function by producing white blood cells (components of blood) and antibodies. The toxins or harmful microorganisms are identified as foreign substances, and then the cells and antibodies produced by the lymph nodes or lymphoid tissues kill or destroy these substances. Vaccines delivered using the gene transfer technology enhance the humoral response, which involves the secretion of antibodies to the antigens (foreign material) by B lymphocytes (white blood cells) in the injected individual, which may allow the individual's immune system to fight the tumors and prevent the complications related to the cancer that may be fatal.
Skin disease: A gene delivery technique for vaccination of stress protein, i.e., heat shock protein (HSP) 70, is being studied to understand its role in increasing the skin's pigment in vitiligo. Vitiligo is a chronic skin disease characterized by a loss of pigment in the skin, leading to formation of irregular white patches. Proteins are organic (carbon-containing) compounds composed of amino acids. This research might help in developing treatment strategies against the skin disease; further research is being conducted in this area.
Viruses: Gene transfer technology is also being used to study the effectiveness of vaccines targeted against certain human viruses, such as orthopoxvirus, (a genus of poxviruses, including smallpox), which commonly affects mammals, and the flu virus. A genus is a group of species with similar characteristics. Researchers are conducting studies to immunize mice against the flu virus with a DNA vaccine using gene transfer technology.
Implications
DNA vaccines: Gene transfer techniques have been used as one of the methods for DNA vaccination, which induces humoral and cellular immune responses. Humoral immunity involves secretion of antibodies in response to antigens by 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 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 is possible due to the exposure of antigens to the host's immune system in a natural form, similar to that achieved with live attenuated vaccines.
Farming: Gene transfer technology may be used for various reasons to alter (genetically modify) the genetic material found in plants. Many foods in the United States, including corn, soybeans, and canola, are genetically modified using various techniques. 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 nonmodified 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 DNA transfer methods, plants, including trees, have been genetically modified to help reduce groundwater pollution. These plants are designed to reduce the amount of heavy metal pollution in contaminated soil, thereby reducing pollution.
Crops such as rice, corn, soybeans, sweet potatoes, apples, tomatoes, cantaloupes, and other fruits and vegetables, have been genetically modified using DNA delivery methods. This has been done to improve taste, color, size, and overall quality; to reduce maturation time; to increase nutritional value; to increase tolerance to extreme temperatures; and to improve resistance to diseases, pests, and herbicides.
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 these techniques to improve the animals' health and feed efficiency.
Therapeutic applications: Gene transfer technology may be used to introduce genes that will provide instructions for making proteins that might be therapeutic. For example, genes that produce clotting factors (substances that help in clotting of blood) can be introduced into individuals with bleeding disorders, or genes that increase the production of red blood cells (RBCs) could be introduced into individuals with anemia. Anemia refers to a condition with decreased RBCs in the blood or decreased hemoglobin, i.e., the protein component of RBCs that helps carry oxygen to tissues throughout the body.
Limitations
Ethical: Some people argue that artificially altering living things is a violation of the natural organisms' basic value and that changing the genetic makeup of a living organism is ethically wrong. As gene transfer techniques gain popularity, new laws will be needed to address the ethical and social issues surrounding genetic alteration.
DNA stability: The stability of altered DNA following gene transfer using gene delivery methods is unknown and questionable. It is possible that use of this technology may have serious consequences on all exposed organisms. In some species, the survival of genetically altered animals is low, and the animals that survived showed substantial tissue damage. There is also the question of whether the transferred genes will be inherited normally by offspring. Therefore, the long-term effects of such a technology have yet to be evaluated.
Future research
Intranasal vaccines: Researchers are conducting studies using DNA delivery vehicles to deliver vaccines through the nose (intranasally). Traditionally, vaccines contain small amounts of disease-causing organisms, which allow the immune system to produce antibodies to the foreign invader; consequently, individuals become immune to the specific illness after receiving a vaccine. Although the initial results against human immunodeficiency virus type 1 (HIV 1) antigens show promise, further research is required to prove the effectiveness of intranasally delivered vaccines.
Heart disease: Researchers have conducted studies examining the usefulness of directly injecting DNA plasmids, which are structures in cells that consist of DNA and which can exist and multiply independent of the chromosomes (long pieces of DNA carrying genetic information found in the center (nucleus) of the cell) into the heart. Although many complications have been reported, this technique has the potential to be effective in treating or preventing heart disease in the future.
Liver: Scientists are studying several gene transfer techniques to treat liver disorders, as a potential alternative to hydrodynamics-based transfection, which is a method that involves the injection of DNA through the veins. Transfection refers to the transfer of DNA or other agents, such as drugs, by gene transfer methods into eukaryotic cells that are used in mammalian cells (e.g., human cells) Although certain complications, such as the death of liver cells, have been reported in some studies, gene transfer technology with a few modifications has the potential to be used as gene therapy for liver diseases.
Cancer: Several studies are being conducted to use gene transfer as a strategy to deliver DNA vaccines directly into the skin. This approach, which uses gene gun technology, enhances the potency of the vaccine, due to an efficient DNA delivery mechanism; it might be especially useful in treating tumors (cancerous growths). Gene gun technique is a process of introducing DNA or RNA into living tissues by coating microscopic gold or titanium particles (bullets) with the nucleic acid and using a gene gun to force the particles into the skin of the organism. Synthetic polymer particles like polymethyl methacrylate (PMMA) have the potential to initiate immune responses by producing inflammatory cytokines, which are a type of protein used in cellular communication. Polymers are multiple small molecules of the same substance. Some studies have indicated that these particles cause protective activity against certain cancers. Hence, scientists are evaluating gene transfer technology for the delivery of PMMA particles mixed with plasmid DNA (i.e., DNA vaccination) to treat cervical cancer, which affects the cervix, the lower part of the womb or uterus.
Diolistics: Diolistics is a rapid and efficient technique that is used for the identification of the structure and function of the cells in tissues and for understanding the cellular mechanisms in the body. This technique involves incorporation of fluorescent dyes (e.g., carbocyanine) into cells with the help of gene delivery methods. Once the dye is injected into the cells, it becomes attached to the cell structures, which can be seen within minutes. This process may last for certain time, depending on factors such as the type of the dye used, type of the cell or tissue into which the dye is injected, and the gene delivery method. Scientists are using diolistics to study the structure and function of nerve cells and neuronal genes, which may help in the identification of genes that cause certain nervous system disorders and their processes. Further research is required in this area so as to develop more effective treatments for such conditions.
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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