Defination And Introduction To The Transgenic System:
Nowadays, breakthroughs in molecular biology are happening at an unprecedented rate. One of them is the ability to engineer transgenic animals. The term "transgenics" refers to the science of inserting a foreign gene into an organism's genome. An animal is "transgenic" once a scientist inserts DNA from another organism. This process allows scientists to transfer beneficial genes from a different animal, bacterium, or plant.
Defining The Term - Transgenic Animal:
There are various definitions for the term transgenic animal.
A transgenic animal is one whose genome has been changed to carry genes from other species. [1]
Transgenic animals are animals which have been genetically transformed by splicing and inserting foreign animal or human genes into their chromosomes.
The term transgenic animal refers to an animal in which there has been a deliberate modification of the genome - the material responsible for inherited characteristics - in contrast to spontaneous mutationExamples Of Transgenic Animals:
The first transgenic animal, a mouse, was produced in 1981. In an effort to determine which genes were involved with cancer, a gene was inserted into the mouse that made it susceptible to cancer.
In 1985, the first transgenic farm mammal was produced, a sheep called "Tracy". Tracy had a human gene that expressed high levels of the human protein alpha-1-antitrypsin. The protein, when missing in humans, can lead to a rare form of emphysema.
Many more animal clones have been generated in the mean time. For example, cloned cows appeared in 1999 and now there are cloned pigs that have been modified to reduce transplant rejection of pig organs in humans. Cloned pets (cats and dogs) have been created too. There are even cloned mules.
Harvard scientists made a major scientific breakthrough when they received a U.S. patent (the company DuPont holds exclusive rights to its use) for a genetically engineered mouse, called OncoMouse® or the Harvard mouse, carrying a gene that promotes the development of various human cancers. [1]
GSK scientists engineered the overexpression of the human mitochondrial transporter protein, "uncoupling protein-3" (UCP-3), in skeletal muscle in mice. In this model, the transgenic mice were found to eat more than wild-type littermates, yet remain leaner and lighter. The mice also exhibit lower glucose and insulin levels and an increased glucose clearance rate, leading to the hypothesis that compounds that regulate expression of UCP-3 might be of use in treating obesity. [5]
In theory, large quantities of the human protein can be produced in the animal's milk and subsequently purified for use in medical therapies. It has been gaining application among biotechnologists since the development of transgenic "super mice" in 1982 and the development of the first mice to produce a human drug, tPA (tissue plasminogen activator to treat blood clots), in 1987.
In 1997, the first transgenic cow, Rosie, produced human protein-enriched milk at 2.4 grams per litre. This transgenic milk is a more nutritionally balanced product than natural bovine milk and could be given to babies or the elderly with special nutritional or digestive needs. Rosie's milk contains the human gene alpha-lactalbumin. [1]
The A. I. Virtanen Institute in Finland produced a calf with a gene that makes the substance that promotes the growth of red cells in humans. [1]
In 2001, two scientists at Nexia Biotechnologies in Canada spliced spider genes into the cells of lactating goats. The goats began to manufacture silk along with their milk and secrete tiny silk strands from their body by the bucketful. By extracting polymer strands from the milk and weaving them into thread, the scientists can create a light, tough, flexible material that could be used in such applications as military uniforms, medical microsutures, and tennis racket strings. [1]
Advantages:
The benefits of transgenic animals include:
· Large-scale, low-cost production independent of proximity to the oceans and with reduced environmental impacts;
·Increased growth rates;
·Improved disease resistance;
·Improved food-conversion rates;
·Leaner meat;
·Increased muscle mass;
·Improved wool quality;
·Improved nutritional quality or appeal; and
·More efficient use of indoor water-recycling plants.
Specifically, the environmental and nutritional benefits resulting from aquaculture outweigh potential risks, which include the runoff of pollutants into closed or semi-closed waterways; the elimination of coastal forests and ecosystems; the potential for increases in waterborne disease and parasites; and sustainability. [8]
Advantages Over Selective Breeding:
Transgenic technology is an extension of agricultural practices that have been used for centuries: selective breeding and special feeding or fertilizing programs. It may reduce or even replace the large-scale use of pesticides and long-lasting herbicides. Transgenic technology is still experimental and is still very expensive. However, it offers a number of advantages over traditional methods.
Compared with traditional methods, transgenic breeding is:
More specific – scientists can choose with greater accuracy the trait they want to establish. The number of additional unwanted traits can be kept to a minimum.
Faster – establishing the trait takes only one generation compared with the many generations often needed for traditional selective breeding, where much is left to chance.
More flexible – traits that would otherwise be unavailable in some animals or plants may be achievable using transgenic methods. Less costly – much of the cost and labour involved in administering feed supplements and chemical treatments to animals and crops could be avoided.
Environmentally friendly – allowing less use of chemical pesticides and herbicides and reduced tillage leading to less land degradation.
Overall, the use of transgenic technology has many advantages over traditional methods. Transgenic breeding is said to be more specific, faster, and less costly. Right now research is limited to traits involving one or a few genes. Before scientists can manipulate complex traits, there is going to be the need for many years of research.
Applications Of The Transgenic Animal
Research into transgenic animals could prove useful in several ways. Scientists can provide animals with beneficial genes or traits, such as disease resistance, that will improve their quality of life and bolster waning populations. Transgenic animals may also be designed for organ production, helping to ease the critical shortage of kidneys and livers available for transplants. In addition, scientists are researching ways to produce proteins or drugs in transgenic animals.
Applications of transgenic animals are described in detail in this section, which can be categorized in three groups:
Medicinal Applications:
1. Models of human disease processes:
One of the most important applications of transgenic animals is the development of new animal models for human disease. Gene targeting is being exploited by scientists to create models of human disease. The genetic setup of an animal may be modified in such a way that it develops a disease similar to an equivalent human disease.
Hundreds of transgenic rodent lines have been produced by introducing into the genome genetic sequences such as viral transactivating genes and activated oncogenes implicated in specific pathologies. Transgenic rodent models have been characterized for several human diseases including cardio-vascular disease (Walsh et al., 1990), cancer (Sinn et al., 1987), autoimmune disease (Hammer et al., 1990), AIDS (Vogel et al., 1988), sickle cell anemia (Ryan et al., 1990), muscular dystrophy, Lou Gehring's disease, and neurological disease.
Here are some examples of transgenic animals developed as models of human disease:
Transgenic animals can serve as models for many malignant tumors. Inserting the c-myc oncogene, which regulates cell growth, into a mouse creates a transgenic strain with a high rate of spontaneous tumors. The type of tumor depends on the promoter placed in front of the c-myc gene in the contruct. The mammary tumor virus (MTV) promotor increases the incidence of breast adenocarcinomas. The immunoglobulin heavy-chain enhancer (IgH), when inserted along with the c-myc, results in a strain of mice with a high incidence of lymphoblastic lymphomas. Although mice have been the most frequent hosts for transgenic modification, other domestic animals have also been used.
Transgenic mice overexpressing the amyloid precursor protein form deposits in the brain that resemble the amyloid plaques found in Alzheimer's patients. Mouse models such as these can potentially be used to test drug therapies and to learn more about the progression of the disease.
Harvard scientists made a major scientific breakthrough when they received a U.S. patent (the company DuPont holds exclusive rights to its use) for a genetically engineered mouse, called OncoMouse® or the Harvard mouse, carrying a gene that promotes the development of various human cancers. [1]
Transgenic animals enable scientists to understand the role of genes in specific diseases. By either introducing or inactivating particular genes, researchers can often for the first time discover the root causes of diseases associated with gene defects. For example, GSK scientists engineered the overexpression of the human mitochondrial transporter protein, "uncoupling protein-3" (UCP-3), in skeletal muscle in mice. In this model, the transgenic mice were found to eat more than wild-type littermates, yet remain leaner and lighter. The mice also exhibit lower glucose and insulin levels and an increased glucose clearance rate, leading to the hypothesis that compounds that regulate expression of UCP-3 might be of use in treating obesity. [5]
Reasons for using the transgenic animal as a model for human diseases:
Transgenics may spare the use of higher animals. The creation of transgenic animals is resulting in a shift from the use of higher order species to lower order species. In the long term, a reduction in the number of animals used, for example to study human diseases is possible due to a greater specificity of the transgenic models developed. This shift in the patterns of animal use is being monitored by the CCAC through the use of the Animal Use Data Form. An example of the replacement of higher species by lower species is the possibility to develop disease models in mice rather than using dogs or non-human primates. [2] On the other hand, the success of the method has led to using its potential for investigating a wider range of diseases and conditions. The actual use of some species may be increased. [5]
2.As organ transplant donors to humans:
Another rapidly-moving field is the potential use of transgenic pigs for use as organ transplant donors to humans. Patients die every year for lack of a replacement heart, liver, or kidney. For example, about 5,000 organs are needed each year in the United Kingdom alone. Transgenic pigs may provide the transplant organs needed to alleviate the shortfall. Currently, xenotransplantation is hampered by a pig protein that can cause donor rejection but research is underway to remove the pig protein and replace it with a human protein. [1]This kind of research is still very much in its infancy. If successful, however, this research could transform the lives of the many patients awaiting organ transplants.
Transgenic animals are being developed by some companies to provide new organs for transplantation such as kidneys, livers and hearts. Transgenic pigs with human histo-compatibility genes have been bred in the hope that their "humanized" organs will not be rejected by a patient's immune system. [10]
3. Proteins of medical importance to humans:
One important application of transgenic technology is the generation of transgenic livestock as "bioreactors." Transgenic animals can produce biological products. It may be possible to use transgenic animals to make rare biological products for medical treatment. Milk-producing transgenic animals are especially useful for medicines. Key human genes have been introduced into sheep, cows, goats, and pigs so that the human protein is secreted into the milk of the transgenic animal. In theory, large quantities of the human protein can be produced in the animal's milk and subsequently purified for use in medical therapies. [15]
Principle behind the technique:
The major function of the mammary gland is to produce proteins. The mammary gland is capable of producing milk that carries over 40g/L of protein. Advantage of the unique properties of this "natural protein secretion organ" is taken in this technique. By utilizing molecular biology technology, we can design DNA constructs that reliably express high levels of therapeutic proteins in the milk of the animals that carry the transgene. Advantage of the normal mammalian protein processing mechanisms is taken to synthesize properly folded and assembled complex proteins. Although the epithelial cells in the mammary gland do not usually express antibodies, it has been found that the machinery needed to properly fold and assemble the heavy and light chains of antibodies are well represented in these cells. By utilizing the milk specific promoters to express the heavy and light chains, the cellular machinery is capable of secreting high levels of properly folded antibody.
Since this is a mammalian cell system, it is capable of post-translational modifications such as glycosylation and gamma carboxylation. Many recombinant proteins, most of which are of human origin, require glycosylation for proper function or pharmokinetics. This system provides high level expression combined with mammalian modifications-unique to production systems. This method permits flexible scale-up of protein manufacturing to meet increasing production needs throughout the product development process. Scale-up is as simple as breeding more transgenic animals. This is easier and less expensive than building and validating a larger biopharmaceutical fermentation or mammalian cell culture facility, therefore reducing overall capital costs.
Examples:
An early example of this technology by John Clark and colleagues was the production of transgenic sheep expressing the human blood-clotting factor IX needed by many patients with hemophilia. These researchers placed the human factor IX gene under the control of a piece of sheep DNA that normally turns on the beta-lactoglobulin gene in the mammary tissue. Though the sheep secreted factor IX into their milk, the levels of the protein were very small. With advances in the efficiency of creating and expressing genes in transgenic farm animals, therapeutic proteins can now be isolated. [15]
In 1997, the first transgenic cow, Rosie, produced human protein-enriched milk at 2.4 grams per litre. This transgenic milk is a more nutritionally balanced product than natural bovine milk and could be given to babies or the elderly with special nutritional or digestive needs. Rosie's milk contains the human gene alpha-lactalbumin. [1]
Human alpha-1-antitrypsin, a protein used to treat the rare genetic disorder of alpha-1-antitrypsin deficiency, is also produced by this technique. [1]
Other human proteins that have been expressed in transgenic animals include: anti-thrombin III (to treat intravascular coagulation), collagen (to treat burns and bone fractures), fibrinogen (used for burns and after surgery), human fertility hormones, human hemoglobin, human serum albumin (for surgery, trauma, and burns), lactoferrin (found in mother milk), tissue plasminogen activator, and particular monoclonal antibodies (including one that is effective against a particular colon cancer). Animals mostly used for this work are pigs, cows, sheep, and goats. [3]
Comparison with other methods of protein production:
There are four other means of commercial protein production. E. coli production, which was the first commercialized, is very efficient, but limited to simple non-glycosylated proteins. Although the cost of production is low, the cost of processing and refolding the proteins is significant.
Fungal systems, such as Pichia or filamentous fungi allow efficient production of some secreted proteins, but the glycosylation is usually high mannose which can affect the pharmokinetics of the protein.
There is also the baculovirus production system, which can produce a wide range of proteins in small scale, but has yet to be scaled up to commercial levels.
The standard method for producing complex glycosylated proteins, (i.e. Monoclonal Antibodies) is with cell tissue culture. The protein may be properly folded and modified, but the low yields per cost of production facility limit the number of proteins that can be developed.
Recombinant protein concentrations in the milk of transgenic animals are substantially higher than levels attained in cell tissue cultures. Expression levels of 2 to 10 grams of recombinant protein per liter of milk are readily achievable in transgenic livestock. In comparison, highly optimized cell cultures can typically generate 0.2 to 1 gram per liter of culture medium. It appears that transgenic technology can achieve the high levels of recombinant protein production normally found only in prokaryotic systems. It has the added benefit in that it is a mammalian system that can secrete complex, glycosylated proteins, similar to tissue culture. Thus it has the best of both technologies, with the added advantage of lower capital cost for the production facility.
Transgenic production takes advantage of normal mammalian protein processing mechanisms to synthesize properly folded and assembled complex proteins -- all within the cells of the mammary gland. This method permits flexible scale-up of protein manufacturing to meet increasing production needs throughout the product development process. Scale-up is as simple as breeding more transgenic animals. This is easier and less expensive than building and validating a larger biopharmaceutical fermentation or mammalian cell culture facility, therefore reducing overall capital costs.
Recombinant protein concentrations in the milk of transgenic animals are substantially higher than levels attained in cultures of yeast, bacteria, insect cells or mammalian cells. Expression levels of 2 to 10 grams of recombinant protein per liter of milk are readily achievable in transgenic livestock. In comparison, highly optimized cell cultures can typically generate 0.2 to 1 gram per liter of culture medium.
Animal most commonly used for the purpose of protein production:
In choosing a species of animal it is optimal to have animals that have been bred for significant milk production and also have a relative short generation time. Formally, choices range from mice, one of the model systems with a generation time of 3 months and milk yield of 1 ml, to rabbits with an 8 month generation time and 4 liter yield, to the largest commercial species, the cow. Cows have a generation time of 3 years, with an annual milk yield of 8000 liters.
Since time is critical, goats are a logical alternative with a generation time of 18 months and a yield of nearly 800 liters. As a dairy breed, goats show efficiency of milk production that is unrivaled. As a dairy production animal goats are utilized all over the world. Significant expression, (2-10g/L) of recombinant proteins in lactating goats has been shown. With an annual yield of 800 liters, over 1 kilogram of recombinant protein can be produced per lactating animal. The scale-up of the goat herd following standard breeding is straight-forward and the production of 100's of kilograms of recombinant proteins can be readily achieved. This is well within the levels expected for most recombinant protein markets. Their dairy characteristics combined with their relative short generation time allow meeting our goals. Small amounts of the recombinant protein for initial testing can be delivered within a year, followed by high levels during normal lactation. By utilizing a known dairy animal, the scale-up for large volume production is straightforward. Goats are an ideal dairy species as produce large volumes of milk with high protein content, and are generally accepted as a source of dietary milk. They are relatively easy to breed and maintain. Goat milk has been extensively characterized biochemically, and this makes it more straightforward to develop protein purification procedures.
4. To test the safety of new medicines and vaccines:
Because transgenic models can highlight specific characteristics such as certain mechanisms involved in the formation of tumors, they can demonstrate more clearly the possible side effects of new therapies. Their use in early toxicity trials may also serve to prevent the subsequent use of a larger number of animals in the development phase. Toxicity-sensitive transgenic animals have been produced for chemical safety testing.
Transgenic animals can also be used to test the identity and purity of human proteins used as drugs. A transgenic animal that makes a human protein (e g human insulin) will recognise this substance as its own and will therefore not produce an immune response against it. As a consequence, the identity and purity of the product can be tested more efficiently in such animals, thereby saving the use of many laboratory animals otherwise needed to obtain a statistically significant result. [13]
5. Genatic Research
Genetic models to study the effects of genetic changes on development:
Frequently used in genetic research are transgenic fruit flies (Drosophila melanogaster) as genetic models to study the effects of genetic changes on development. Flies are often preferred over other animals for ease of culture, and also because the fly genome is somewhat simpler than that of vertebrates. Transgenic mice are often used to study cellular and tissue specific responses to the disease. [15]
Agricultural Applications:
6. Disease resistance:
Scientists are attempting to produce disease-resistant animals, such as influenza-resistant pigs, but a very limited number of genes are currently known to be responsible for resistance to diseases in farm animals. [1]
7. Production of milk low in cholesterol:
Transgenic cows exist that produce more milk or milk with less lactose or cholesterol. [1]
8. The polled (hornless) condition in cattle. [10]
Industrial Applications:
9. material fabrication:
In 2001, two scientists at Nexia Biotechnologies in Canada spliced spider genes into the cells of lactating goats. The goats began to manufacture silk along with their milk and secrete tiny silk strands from their body by the bucketful. By extracting polymer strands from the milk and weaving them into thread, the scientists can create a light, tough, flexible material that could be used in such applications as military uniforms, medical microsutures, and tennis racket strings. [1]
10.Increased Meat Production:
An abnormally high quantity of growth hormone in the transgenic animal is responsible for increased meat production. Pigs and cattle that have more meat on them can be produced. In the past, farmers used growth hormones to spur the development of animals but this technique was problematic, especially since residue of the hormones remained in the animal product. [1]
11. Sheep that grow more wool:
Breeding transgenic sheep that grow better wool without needing dietary supplements of sulphur-containing amino acids is under research.
The Future:
Research is presently limited to traits involving one or a few genes. It will probably require many years of research before scientists can manipulate complex traits (such as meat quality or animal behaviour) that are influenced by many genes.
Much current research focuses on the understanding and developing useful promoter sequences to control transgenes and establishing more precise ways to insert and place the transgene in the recipient. Much still needs to be done to improve our knowledge of specific genes and their actions and of the potential side effects of adding foreign DNA and of manipulating genes within an organism.
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