Thursday, May 21, 2009

What is Transgenic Locus

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Gene constructs used in genetic transformation posses a promoter, coding region, and termination sequence. In the vicillin promoter, specific for expression in seeds, drives the expression of the gene UDP 6-glucose dehydrogenase in the antisense orientation. The construct also possesses the NOS (noplaine synthase) termination sequence, which marks the site for the end of transcription. Besides the transgene of interest, in general, reporter genes are introduced simultaneously to facilitate the identification and selection of transformed individuals.

For the selection of the transformed cells, the gene construct contains a gene sequence that codes for antibiotic or herbicide resistance. Frequently, neomycin or hygromycin antibiotic resistance genes or the phosphinotricim acetyl transferase herbicide tolerance gene is included under a strong constitutive promoter, such as 35SCaMV. The transformed cells would be the only ones possessing the capability to grow in a medium with a selective agent (antibiotic or herbicide), thereby facilitating their selection.

Frequently, a gene reporter is also included in the genetic construction. The function of this reporter is to allow the visual identification of transformed cells. Three genes have been used as reporters in plant transformation: Glucaronidase (GUS), Luciferase (LUC) and Green Fluorescent Protein (GFP). GUS allows the identification of transformed individuals by the expression of a blue color, because they become blue in the presence of the chemicals X-Gal and IPTG (isopropyl beta D-thiogalactoside). Luciferase, a protein in fireflies, turns the transformed individuals phosphorescent, and GFP, isolated from a species of jellyfish, codes for a fluorescent-green color in transformed individuals.

Genetic transformation of individuals is a difficult task. The science behind the methods is understandable on a basic level, but the results from the procedures do not always work out as planned. Specific gene sequences are needed to induce the expression of a transgene, and genes are needed to identify the transformed cells. Still, the use of transformation is being improved to more accurately express desired traits in different organisms. The comprehension of the intricacies of transformation is a key to understanding the broad applications of biotechnology.


Tags: Bio Technology, Bio Genetics, Genetic Transformation

Know the Gene Expression

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All cells possess the typical number of chromosomes of their species. Therefore, root, epidermis, or pod cells of a soybean plant possess all 40 chromosomes typical of this species. However, not all of the genes are expressed in every cell. For instance, genes that code for chlorophyll production are expressed in the leaves and any other green part of the plant. However they are silenced in the roots, which is the reason these cells do not contain chlorophyll. Gene regulation is a complex process that is affected by a series of factors. A common occurrence in genetic engineering is a lack of expression after a gene has been transformed into an organism. Therefore, an understanding of mechanisms involved with gene expression is critical in genetic transformation.

In bacteria, some genes are activated while others are silenced, depending on the conditions in which these microorganisms are grown. For example, the bacteria Escherichia coli can use two different carbohydrates, lactose and glucose, as energy sources. The bacteria needs to synthesize specific enzymes that catalyze the breakdown of the carbohydrates into energy. The enzymes, like all other proteins, are coded by genes. When E. coli is cultivated in a medium with both glucose and lactose (preferably glucose), it metabolizes. The genes coding for the production of the enzymes that metabolize glucose are thus expressed preferentially. The metabolism of lactose requires an additional enzyme that is only synthesized, or activated, after the medium runs out of glucose and lactose is the only energy source available. This phenomenon is called gene regulation.

Gene expression in more complex organisms is still not completely understood. The complexity of gene regulation is a puzzle in the zygote, a cell formed by the union of sperm and egg cells, in which the genes coding for differing functions have to be activated in a precise and orderly manner. The same genetic information present in the zygote is also present in any other cell in the body, from muscles to skin. Obviously, different genes are activated or expressed in each organ in a different way.

Gene expression is not just a function of where the cell is, but also the result of environmental stimuli. Cells of a floral bud of soybeans differentiate into flowers when the plant is grown during long nights. If the soybean plant is grown during short nights, it continues vegetative growth and does not bloom. Another example of gene regulation occurs with animals, including humans. Testicle and ovary cells do not start the production of sexual hormones until the individual reaches puberty.

Another example of the complexity and importance of gene regulation can be observed in the metamorphosis and development of butterflies and moths. These insects take three forms during their lives: caterpillar, pupa, and adult butterfly or moth. The insect possesses the same genes and DNA during these three different developmental phases. Although the caterpillar, pupa, and adult have the same genes, it is interesting to observe that different genes are expressed in the three developmental phases. In the caterpillar phase, the genes for production of several legs and a stronger mouth capable of chewing leaves are expressed, but not the genes for production of wings. However, the genes for the formation of a delicate mouth apparatus, appropriate for nectar feeding, and genes for the formation of wings are active in the insect's adult phase. The gene expression pattern changes during insect development to allow for the correct progression of its life cycle.

The mechanisms regulating gene expression involve regulatory genes. As opposed to the genes discussed up to this point, these DNA sequences do not code for any protein. Their function is to promote the activation or the silencing of genes.

An important part of gene regulation is the promoters. A promoter is a DNA sequence preceding the gene, which contains regulatory sequences to control the rate of RNA transcription. Promoters control when and in which cells a certain gene is expressed. Through the manipulation of promoters it is possible to induce superexpression, underexpression, or even gene silencing.

Some promoters are constitutive—that is, they induce gene expression continually—whereas others are inducible. Among these, there are some that are chemically inducible, and others are activated by heat, light, or hormones. Some promoters are active in certain tissues and organs, but not in others. In this case, they are considered tissue-specific promoters, as in the case of chlorophyll production. The promoters of the chlorophyll genes are not active in roots, but they are active in the leaves and in all green parts of plants.
Some of the promoters frequently used in genetic engineering of plants include the following:
1) Constitutive
a) UBI from corn
b) 35SCaMV from a cauliflower virus

2) Tissue-specific
a) Phaseolina promoter, a seed-specific promoter from field beans
b) Vicillin promoter, a seed-specific promoter from peas
c) Glutamine promoter, an endosperm-specific promoter from wheat

3) Inducible
a) Rubisco 5S promoter, inducible by light

Aside from promoters, other genetic factors are important in proper gene expression. Although the genetic code is universal, it is also considered degenerate, as more than a single codon codes for a certain amino acid. Different organisms have acquired the preferential use of specific codons for certain amino acids during evolution; this can also have an impact in gene expression. That was the case of the Bt gene from Bacillus thuringiensis introduced in corn. Initially, the expression of that bacterial gene in corn was low; however, when a transgene was reengineered to favor the preferential use of certain codons by corn, gene expression occurred at normal levels.

Several other factors can affect the expression of transgenes, such as the presence of a peptide signal, the site of its integration in the genome, the number of copies integrated, and transgene rearrangements during the integration process. Integration of transgenes in the host genome, in general, happens at random; that is, it can occur in any chromosome of the cell and it can land in any part of the chromosome. However, most of the transgenic varieties have the transgene inserted close to the ends of the chromosome. Multiple copies of the transgene are typically introgressed together.

Tags: Bio Technology, Bio Genetics, Genetic Transformation

Problems in Genetic Transformation

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Tissue culture has been identified as one of the largest obstacles in the development of transgenic plant products. It is necessary to develop protocols that allow the regeneration of whole individuals from the transformed cells or tissue. One of the difficulties faced by scientists is that regeneration methodologies work well with some, but not all species or germplasm within a species. This severely limits the spectrum of individuals that can be transformed. In many cases, the procedure has been the transfer of the transgene through classical genetics and breeding methods. An example of this is in the genetic transformation of wheat. Genetic transformation of most wheat varieties is very difficult because of problems in tissue culture. One variety, Bobwhite, is the exception, and protocols have been developed for the transformation of this wheat variety. Once a gene has been successfully transferred into Bobwhite, it can be moved into other varieties through traditional breeding methods.

Another difficulty associated with the use of tissue culture in transformation is somaclonal variations. Plants produced from tissue culture have higher mutation rates and the appearance of abnormal variation. This is due to the delicate environment in which cells are cultured. Many times, the cultured plants have problems associated with the cell cultures and not from the transgene integration.

Transformation methods currently in development promise to revolutionize the introduction of genes in plants. Some of these methods are already being used with the model plant Arabidopsis thaliana, commonly known as mouse ear cress. One of the methods involves the submersion of floral buds in a solution containing plasmids bearing the transgenes. Another alternative technique, still in development, is the transformation of seeds mediated by Agrobacterium tumefaciens. Although the methods have been used with success in Arabidopsis, the literature does not report its use in crop species. The key aspect of these two methods is that transformation is carried out without the need to regenerate plants through tissue culture. These methods are exciting because the transformation procedure works on the seeds that can then be planted to identify transgenic individuals.

Tags: Bio Technology, Bio Genetics, Genetic Transformation

Methods of Genetic Transformation

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Among the several methods of plant transformation, four have yielded the best results: Agrobacterium species-mediated transformation, microprojectile bombardment, microinjection, and direct transformation. Each of these methods has merits and limitations and is used in specific situations. At this time there is no single technique that is suitable for all species.

Agrobacterium Mediated Transformation
Tumors and uncontrolled cellular growth in plants can occur due to genetic factors or bacterial and viral infections. An example is crown gall in plants, where tumors are caused by bacteria that causes uncontrolled growth on the stem of the infected plants. This problem is caused by Agrobacterium tumefaciens, a soil bacterium that infects some plants because of a wound on the plant. Plasmids present in the bacteria are responsible for tumor growth after infection by A. tumefaciens. The bacteria are able to recognize wounds on the plant, and this induces the transfer of the bacterial plasmid into the plant. The plasmids are capable of integrating into the DNA of the host plant, causing uncontrolled plant growth and the formation of tumors. The ability of A. tumefaciens to efficiently transfer plasmid DNA into the host has made it important in early studies in genetic transformation.

Agrobacterium tumefaciens was the first vector used for introduction of foreign DNA in plant cells. Although Agrobacterium has only been used to infect dicot plant species, such as soybean, tomato, pea, and cotton, the protocol has been modified to allow the bacteria to infect some monocot (grass) species as well. Many research groups working with plants have found this to be the preferred transformation approach. Another soil bacteria, Agrobacterium rhizogenes, causes the growth of secondary roots after infection. This bacterial species has also been used for plant transformation.

The basis of this transformation method is the bacterial plasmid, which contains the genetic sequence that is integrated into the host genome. One of the most important parts of a plasmid is the region responsible for the translocation of its DNA into the host plant genome. This is called transfer DNA (T-DNA), and this area of DNA is key to the tumor growth in infected plants. The region is located between the right border and left border (RB and LB) of the plasmid. Plasmids also contain other important DNA sequences; some of them control the production of auxin and cytokinin, two important plant hormones involved in tumor formation. With the use of the restriction enzymes, a transgene can be introduced between the right border and left border of the plasmid, allowing the bacteria to transfer novel genes into the recipient plant.

One of the techniques used for transformation mediated by A. tumefaciens uses leaf disks. Leaf disks of about 6 mm in diameter are cultured on a tissue-culture media containing A. tumefaciens with plasmids containing the transgene. After approximately a month of incubation in the tissue culture medium, seedlings start to develop on the leaf disks. Through selection methods, transgenic seedlings are identified for whole plant regeneration.

Microparticle Bombardment
This technique has also been called microprojectile acceleration or biolistics, but microparticle bombardment is the formal name for the machine called a gene gun. This method, developed at Cornell University, was designated biolistic (biologic + ballistics = biolistic), because high-speed microscopic projectiles (microprojectiles) are accelerated into the cells to be transformed.
This transformation method consists of the acceleration of a macroprojectile loaded with millions of tungsten or gold microspheres about 1 ┬Ám in diameter (microparticle). The microspheres are coated with the transgene, or DNA of the gene of interest. Microspheres have a high specific mass, allowing them to acquire the needed momentum to penetrate the target cells. The macroparticle is propelled in the direction of the cells at high speed, but it is retained, after a small distance, on a steel mesh so that the microparticles continue in the direction of the target cells. Helium gas at high pressure is used to propel the macroparticle, and the acceleration chamber operates under a partial vacuum, which allows for improved microsphere movement. Once inside the target cells, the DNA coating the microspheres is released and can be integrated into the plant's genome.

Many of the commercial transgenic crop varieties on the market today were developed using the gene gun. However, due to its cost and the complex integration patterns resulting from this method, several research groups are reducing its use.

Microinjection
This method was developed for animal transformation but has also been extended to plants. Although very difficult and laborious, DNA microinjection has yielded positive results and has been used in several laboratories.

In this technique, microcapillary needles are used to introduce DNA directly into cells. Each cell to be transformed must be manipulated individually. One of the advantages of this method is that the optimum amount of DNA can be injected into the target cells, which helps to ensure optimal integration. Positive results have already been obtained in several crop species such as corn, wheat, soybean, tobacco, and rice, and in animals like salmon, cattle, and swine.

Direct Transformation
Transformation using direct methods was accomplished soon after the first Agrobacterium-mediated transformation. These methods use protoplasts (cells after the removal of the cellular wall) as targets for transformation. This is a simple method that consists of adding great amounts of transgenic plasmids to a protoplast culture, which guarantees that a small proportion of the protoplasts will be taken up (assimilated) by the plasmids. The assimilation rate can be increased with the addition of polyethylene glycol (PEG) or the use of an electric discharge (electroporation). No barrier to direct transformation has been detected, indicating that this method can be used with virtually any species. The problem with this method lies in the difficulty of regenerating a whole plant starting from protoplasts. Therefore, it has not been used as widely as the other methods.

Tags: Bio Technology, Bio Genetics, Genetic Transformation

Understanding Genetic Transformation

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The term genetically modified is frequently used to describe organisms that were genetically transformed or engineered. The science of genetic engineering was developed with the objective of building genes for genetic transformation. Genetic transformation systems possess three main components:
1) A mechanism for introduction of the foreign DNA into the target cell.
2) A cell or tissue suitable for transformation.
3) A method for the identification and selection of transformed cells or individuals.

Success in transformation for any species depends on these three components. Obviously, each one must be optimized and, therefore, as technology develops, transformation should become a more routine activity. The final objective in transformation is the introduction of a new trait in an individual. When the desired trait exists in any other sexually compatible individual, the first alternative should be to transfer the trait through crossing and selection, as has been done in conventional breeding since the 19th century. Modern soybean, corn, cotton, and wheat varieties, as well as swine, cattle, and poultry lines used in agriculture to feed the world, were initially obtained by traditional methods of crossing and selection.

One of the main limitations of conventional genetic improvement is that the breeder is limited to traits among species that are sexually compatible. For instance, the field bean is a species rich in sulfur-containing amino acids. However, beans are naturally deficient in lysine. On the other hand, rice is naturally rich in lysine, but deficient in sulfur-containing amino acids. It is not possible to naturally cross these species, so the conventional plant breeder is unable to develop a new field bean variety with elevated lysine levels or a rice cultivar rich in sulfur-containing amino acids. Genetic transformation allows the exchange of genes between organisms previously limited by sexual incompatibility. With genetic engineering and transformation, it is possible to transfer genes among bacteria, animals, plants, and viruses. In fact, one of the areas of research in biotechnology is the improvement of nutritional profiles in crops. New, more nutritional bean and rice varieties can now be developed through advances in genetic engineering.The basic tools for genetic transformation are restriction enzymes, which are used to cut DNA at specific sites, and ligases, which catalyze the joining of DNA fragments. Using the right restriction enzymes, it is possible to cut the circular bacterial plasmid DNA, causing it to linearize. With a ligase, it is possible to add other DNA fragments containing the gene of interest and join them to the linearized plasmid. Under the right conditions, the ends of the plasmid, now with the added DNA fragments, rejoin to create a new circular plasmid with some DNA modifications. The new plasmid can be introduced into certain bacteria through a process called electroporation, and the bacteria can then be used to transfer the transgene to the target species. If the plasmid DNA is integrated into the genome of the recipient species and the transferred genes are expressed, the individual is considered to be transformed or transgenic.

Tags: Bio Technology, Bio Genetics, Genetic Transformation

Understanding Genetic Transformation

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The term genetically modified is frequently used to describe organisms that were genetically transformed or engineered. The science of genetic engineering was developed with the objective of building genes for genetic transformation. Genetic transformation systems possess three main components:

1) A mechanism for introduction of the foreign DNA into the target cell.

2) A cell or tissue suitable for transformation.

3) A method for the identification and selection of transformed cells or individuals.

Success in transformation for any species depends on these three components. Obviously, each one must be optimized and, therefore, as technology develops, transformation should become a more routine activity. The final objective in transformation is the introduction of a new trait in an individual. When the desired trait exists in any other sexually compatible individual, the first alternative should be to transfer the trait through crossing and selection, as has been done in conventional breeding since the 19th century. Modern soybean, corn, cotton, and wheat varieties, as well as swine, cattle, and poultry lines used in agriculture to feed the world, were initially obtained by traditional methods of crossing and selection.

One of the main limitations of conventional genetic improvement is that the breeder is limited to traits among species that are sexually compatible. For instance, the field bean is a species rich in sulfur-containing amino acids. However, beans are naturally deficient in lysine. On the other hand, rice is naturally rich in lysine, but deficient in sulfur-containing amino acids. It is not possible to naturally cross these species, so the conventional plant breeder is unable to develop a new field bean variety with elevated lysine levels or a rice cultivar rich in sulfur-containing amino acids. Genetic transformation allows the exchange of genes between organisms previously limited by sexual incompatibility. With genetic engineering and transformation, it is possible to transfer genes among bacteria, animals, plants, and viruses. In fact, one of the areas of research in biotechnology is the improvement of nutritional profiles in crops. New, more nutritional bean and rice varieties can now be developed through advances in genetic engineering.The basic tools for genetic transformation are restriction enzymes, which are used to cut DNA at specific sites, and ligases, which catalyze the joining of DNA fragments. Using the right restriction enzymes, it is possible to cut the circular bacterial plasmid DNA, causing it to linearize. With a ligase, it is possible to add other DNA fragments containing the gene of interest and join them to the linearized plasmid. Under the right conditions, the ends of the plasmid, now with the added DNA fragments, rejoin to create a new circular plasmid with some DNA modifications. The new plasmid can be introduced into certain bacteria through a process called electroporation, and the bacteria can then be used to transfer the transgene to the target species. If the plasmid DNA is integrated into the genome of the recipient species and the transferred genes are expressed, the individual is considered to be transformed or transgenic.

Tags: Bio Technology, Bio Genetics, Genetic Transformation

Friday, May 8, 2009

Understanding Stem Cell Gene Therapy

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Stem cell therapy or therapeutic cloning does not involve gene therapy itself. However, in the future it might be used in conjunction with gene therapy for regeneration of tissue and organs after they have been treated with corrective genes. Visually, stem cells are not distinguishable from any other cells of the human body. Under a common microscope (magnification 20 to 40 times), those cells can only be observed using special dyes. Visually there is no significant difference in such cells. The real differences exist at the DNA level, where gene expression is amendable to signals influencing protein expression. The cells can differentiate into any of the 220 cell types of the human body (e.g., kidneys, heart, liver, skin, or retina), a phenomenon called pluripotency. At birth, stem cells can be harvested from an individual's bone marrow, fat tissue, and the umbilical cord. Embryonic stem cells are harvested from embryos up to a few days after fertilization.

Another characteristic of stem cells is their capability to grow indefinitely. Whereas the remaining body cells have a biological programming that limits the number of cell divisions they can go through before dying, stem cells can be maintained indefinitely in a petri dish with nutritive media.

Stem cell therapy provides hope for a cure for patients of incurable afflictions such as Parkinson's disease and Alzheimer's disease, and also for people suffering from paralysis resulting from spinal cord injuries.

At first, some opponents speculated that stem cells would be used in nurseries to produce organs such as livers, hearts, and virtually any other body part. However, most organs possess complex structures with ducts and valves, making it impossible to produce them outside of the organism. Stem cells have opened a new avenue for disease treatment. For example, the injection of stem cells into the liver of a patient with cirrhosis or hepatitis could result in new tissue capable of performing its role. Stem cell therapy also has great potential to cure rheumatoid arthritis and some heart diseases. Recent research has found that spine-injured mice suffering from paralysis were able to move their legs following an injection of stem cells.

Some people believe that if human stem cells are as versatile as those of mice, they might be the long sought after fountain of youth. The combination of stem cells with gene therapy might allow rebuilding of new body parts to substitute for old and defective ones. Right now, different procedures are being tested for curing ADA deficiency. Somatic cell gene therapies have the limitation of lasting for only a few months, which in turn requires repeated applications. With the use of stem cells to regenerate healthy bone marrow cells, a permanent cure is expected, as healthy cells have the capability to grow and divide continuously.

Embryonic stem cells, from embryos about four days old, have been at the center of a heated debate due to ethical issues. The main disagreement is whether or not a four-day-old embryo is already a human life. When would an embryo or a fetus reach the status of life? Those that support the use of embryonic stem cells would argue that human life would not begin until about the 14th day after the fertilization, whereas the opposition argues that life begins at conception (i.e., at the moment of the fertilization of the egg by the sperm). For many, the destruction of embryos for the purpose of treating another human being is wrong. Recently, in the United States, the Bush administration broadened the definition of a child eligible for coverage under the Children's Health Insurance Program by classifying a developing fetus as an "unborn child." Many activists are arguing that the Bush administration's proposal demonstrates its commitment to the strategy of undermining a woman's right to choose abortion by ascribing legal rights to embryos.

Tags: Bio Technology, Bio Genetics, Gene Therapy

What are the Risks associated with Gene Therapy

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The first death associated with gene therapy occurred on September 18, 1999, at the University of Pennsylvania. Jesse Gelsinger was participating in a clinical trial, a biomedical experiment for evaluation of safety and efficiency of a therapy for a disease. Gelsinger, who was 18 years old at the time of the treatment, had a deficiency of ornithine transcarboamylase, an important enzyme in the metabolism of ammonia. Patients with this rare metabolic disorder must maintain a low-protein diet and take a series of medicines to avoid ammonia poisoning in the blood stream. The gene therapy Gelsinger took triggered a chain reaction in his immune system, resulting in hepatic and respiratory failure, and consequently, his death four days after being treated.

Since Gelsinger's death, the University of Pennsylvania has been reevaluating all procedures involved in the vector engineering and in the administration of the therapy. No flaw has been found that would explain such an extreme reaction by his immune defense system. Ever since, the public and the FDA, the agency responsible for oversight of clinical trials in the United States, have been more skeptical and doubtful about whether current scientific knowledge is enough to justify further investigations with humans. The credibility of gene therapy was seriously damaged, resulting in a temporary moratorium on human clinical trials.

Another challenge to gene therapy has been its ephemeral benefits to patients. This has been observed in several clinical trials with cystic fibrosis and ADA deficiency patients, whose cure faded after a few months of therapy, and was followed by a return of the disease symptoms. A possible explanation for that is that the genetically modified somatic cells decreased in amount. Because they are already differentiated and possess only a limited capability to multiply, it is expected that after they are gone, the treated organ could become diseased again.

Future of Gene Therapy

Although the idea of gene therapy has been around for only 20 years, the technique has been drawing a great deal of interest and curiosity through the world. The first trials generated great expectations within the scientific community. Although there have been several disappointments, many believe that it is just a matter of time before the technical and scientific details are mastered and the procedures become routine. This research is being advanced worldwide. In fact, Alain Fischer, a medical doctor in Paris, France, reported the complete cure of two children who had a rare immune deficiency condition.

Another promising result from stem cell research has been reported in type-B hemophilia patients at the Children's Hospital in Philadelphia and at Stanford University, where patients treated with gene therapy presented a reduction in the period for blood coagulation. ADA deficiency, a disease caused by a defective gene for the ADA enzyme present on human chromosome 20 has been a focus for gene therapy in many institutions. In one of the cases, several patients treated with the corrective gene were able to reconstitute their immune systems and are living normal lives out of the isolated bubbles that are needed to maintain an environment free from microbes. The patients started to produce a correct ADA enzyme after receiving the gene therapy.

The potential use of this therapy to cure other more complicated diseases, such as cancer and coronary diseases, also seems promising. Gene therapy is still in its infancy, but it is believed that as it matures, it will become an effective treatment for the myriad of genetic diseases that affect humanity.

Tags: Bio Technology, Bio Genetics, Gene Therapy

What are the pointers to Gene Delivery

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Appropriate methods to deliver DNA used in gene therapy are vital, as the targeted tissues must properly receive the appropriate genes. Gene therapy can be carried out using naked DNA delivered directly into the target cells. However, this procedure of introducing isolated DNA molecules has a very low efficiency rate. To increase the efficiency of DNA uptake by the target cells, special vectors have been engineered for gene transfer. Vectors are plasmids or viruses that are used to move recombinant DNA from one cell to another. A retrovirus is a special class of RNA viruses that can insert its nucleic acid into host cells. The viruses possess a gene for production of the reverse transcriptase, an enzyme that transcribes RNA in DNA in the host cell. Adenovirus, retrotransposons, and liposomes are other vectors used for gene transfer in gene therapy. They are all able to transfer and integrate genes into new cells. Retroviruses used in gene therapy are engineered so that any genes that are harmful to man are removed. Corrective genes are then added to replace the removed genes, and the new, modified retrovirus is then introduced into the patient.

One of the challenges for vectors is to survive the patient's immune system so they can transfer the corrective genes from their genome into the patient's cells. In general, the immune system of the human body contains molecules that immobilize viruses or other microorganisms that could infect the organism. Viruses that escape the immune system need to penetrate the cellular membrane, an additional barrier to infection. Finally, the infecting retrovirus must integrate its genome with that of the host, thereby moving the corrective genes into the genome of the infected cell. This integration happens in a random manner. It should occur in an area of DNA that is not essential to the host genome, or a risk of other complications might occur. Furthermore, the introduced gene must be transcribed and expressed for the production of the correct enzyme. With all these processes at the molecular level, gene therapy becomes a very complex procedure.

Another promising strategy, which has been used for the introduction of therapeutic genes in lung cancer treatment, is the direct injection of the corrective genes into the target area. Using this strategy, scientists have injected a drug containing the normal version of the gene, which suppresses cell tumor growth, directly into the patient's cancerous tumor. This technique bypasses the immune system reaction to the invading vector, a problem frequently associated with gene therapy. Many scientists believe that as gene therapy develops, it will be possible in the near future to easily introduce genes into patients through intramuscular injection, especially for cases of anemia, hemophilia, diabetes, and other diseases related to the circulatory system.


Tags: Bio Technology, Bio Genetics, Gene Therapy

Causes of Genetic Defects

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Each human being carries normal as well as some defective genes. Usually, the individual does not become aware of the presence of a defective gene until a disease associated with the gene is manifested in him or her or in a relative. More than 4,000 medical disorders caused by defective genes have been identified, each with varying degrees of seriousness. About 10 percent of the human population will evidence, sooner or later, some type of disorder. Although genes are responsible for predisposition to disease, the environment, diet, and lifestyle can affect the onset of the illness.

An example of a genetic disease is cystic fibrosis, which frequently becomes evident in the first years of life for the child carrying the defective gene. The mutant gene causes the development of cysts and fibrous tissue in the patient's pancreas and the production of thick and viscous lung mucous. The mucous makes breathing very difficult and, in many cases, is fatal. On average, in Western countries, about 1 child in 2,500 has the disease. If the child receives two defective recessive alleles of the gene named CF (one from each parent), he or she will develop the disease. Patients with cystic fibrosis can reduce the symptoms of the disease with drugs developed through genetic engineering. A cure for cystic fibrosis may come through gene therapy. One possibility is a genetically engineered virus, carrying the corrective gene, which after being introduced into the patient's lung cells would allow the lungs to function properly. The introduced gene would allow the lung cells to produce a protein that eliminates the mucus.
Most people do not manifest genetic diseases because, most of the time, they are carriers of just a single defective copy of the CF gene. As most of the defective genes are recessive, meaning two copies are needed for expression of the disease, most people do not have the disease. This is the reason for the larger incidence of genetic diseases in children from related parents.

If the defective gene, however, is dominant, the disease is expressed in any people that carry the defective gene. Huntington's Disease, a disorder of the nervous system that usually occurs after the age of 45, is an example of a genetic disease caused by a dominant gene.

Having a defective gene does not make disease development a certainty. Besides the large effect from genetics, the environment is also important to the onset of many illnesses. Diseases such as heart disease do have a genetic component, but are largely dependent on diet and lifestyle. Some genetic diseases also have benefits. A classic example of a genetic disease that has a beneficial effect on human survival is sickle cell anemia. There exists in the human population a defective b-hemoglobin gene and individuals carrying two copies of the defective gene develop sickle cell anemia, a blood problem caused by defective hemoglobin and consequently misshapen red blood cells. The genetic mutation in the defective allele of this disease is a single nucleotide change, from an A in normal genes to a T in the mutant. This single nucleotide mutation results in a mutant b-hemoglobin that possesses the amino acid valine instead of glutamine. The mutant b-hemoglobin has less affinity to oxygen, becoming a poor oxygen transporter in the blood.

However, carriers of a single copy of the defective allele do not have the disease, and they are also resistant to malaria. There is an obvious advantage of carrying a single allele of the defective hemoglobin gene, especially in regions where malaria is endemic, as in tropical regions of Africa.
The first case of gene therapy occurred in 1990, at the NIH in Bethesda, Maryland. On that occasion, a four-year-old patient with a severe immune system deficiency (adenosine deaminase enzyme [ADA] deficiency or bubble-boy disease) received an infusion of white blood cells that had been genetically modified to contain the gene that was absent in his genome. Since then, gene therapy has been studied and experimentally tested for several medical conditions.
Diseases caused by the absence of an enzyme or the presence of an inactive enzyme are potential targets for gene therapy. Cystic fibrosis, ADA deficiency, and many other genetic diseases are among the candidates for gene therapy.

Medical Conditions for Which Gene Therapy Is Being Studied are as under
ADA deficiency > Hemophilia
AIDS > Liver cancer
Asthma > Lung cancer
Brain tumor > Melanoma
Breast cancer > Muscular dystrophy
Colon cancer > Neurodegenerative conditions
Diabetes > Ovarian cancer
Heart diseases > Prostate cancer

Tags: Bio Technology, Bio Genetics, Gene Therapy

Understanding Gene Therapy

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Gene therapy has become an increasingly important topic in science-related news. The basic concept of gene therapy is to introduce a gene with the capacity to cure or prevent the progression of a disease. Gene therapy introduces a normal, functional copy of a gene into a cell in which that gene is defective. Cells, tissue, or even whole individuals (when germ-line cell therapy becomes available) modified by gene therapy are considered to be transgenic or genetically modified. Gene therapy could eventually target the correction of genetic defects, eliminate cancerous cells, prevent cardiovascular diseases, block neurological disorders, and even eliminate infectious pathogens. However, gene therapy should be distinguished from the use of genomics to discover new drugs and diagnosis techniques, although the two are related in some respects. The two main types of gene therapy are somatic cell gene therapy and reproductive or germ-line gene therapy. This chapter also discusses therapeutic cloning, which involves stem cell manipulation for tissue and organ production.

Germ-line cell therapy involves the introduction of corrective genes into reproductive cells (sperm and eggs) or zygotes, with the objective of creating a beneficial genetic change that is transmitted to the offspring. When genes are introduced in a reproductive cell, descendant cells can inherit the genes.

Gene therapy of somatic cells, those not directly related to reproduction, results in changes that are not transmitted to offspring. An example of gene therapy in somatic cells is the introduction of genes in an organ or tissue to induce the production of an enzyme. This alteration does not affect the individual's genetic makeup as a whole and it is not transmitted to its descendants. With somatic cell gene therapy, a disabled organ is better able to function normally. This technology has many applications to human health. One variant of somatic cell gene therapy is DNA vaccines, which allow cells of the immune system to fight certain diseases in a method similar to conventional vaccines.

Stem cell therapy involves the use of pluripotent cells, or cells that can differentiate into any other cell type. Stem cells are found in developing embryos and in some tissues of adult individuals. This therapy is similar to a conventional transplant, with the objective of regenerating or repairing a damaged organ or tissue. The procedure has a reduced probability of rejection because it uses the individual's own cells. For instance, stem cells differentiated into nerve cells could be used by patients suffering from paralysis, with the goal of helping them recovering movement; or in cases of heart stroke, muscle cells might be used to rejuvenate the cardiac muscles. Furthermore, the future may bring the growth of stem cells from an individual's body to produce certain tissues or organs in vitro. Stem cell research could eventually blend gene therapy with genetic engineering to create healthy stem cells that can be used to generate healthy organs and tissue.

A fundamental requirement for gene therapy is the correct identification of genes coding for diseases. This can be accomplished at a spectacular speed with the information from the Human Genome Project. Scientific magazines have been announcing, with great frequency, the discovery of genes responsible for several medical conditions, from Alzheimer's disease to baldness. The knowledge of the genes involved in these traits allows unequivocal diagnosis of the disease in the patient, an essential step before treatment can be initiated for the genetic disease. Biotechnology is contributing to the development of the needed genetic tests for detection of defective genes.
The most complex phase in gene therapy is the development of mechanisms to deliver the therapeutic genes to the target organ in an accurate, controlled, and effective way. That step has been developing more slowly and is currently the most limiting factor for gene therapy.

Tags: Bio Technology, Bio Genetics, Gene Therapy