Gene Therapy, Cloning, Agriculture and Bioremediation

Gene therapy

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• Gene therapy using an Adenovirus vector. A new gene is inserted into an adenovirus vector, which is used to introduce the modified DNA into a human cell. If the treatment is successful, the new gene will make a functional protein.
  
• Gene therapy may be used for treating, or even curing, genetic and acquired diseases like cancer and AIDS by using normal genes to supplement or replace defective genes or to bolster a normal function such as immunity. It can be used to target somatic (i.e., body) or germ (i.e., egg and sperm) cells. In somatic gene therapy, the genome of the recipient is changed, but this change is not passed along to the next generation. In contrast, in germline gene therapy, the egg and sperm cells of the parents are changed for the purpose of passing on the changes to their offspring.
  
• There are basically two ways of implementing a gene therapy treatment:
  
• 1. Ex vivo, which means “outside the body” – Cells from the patient’s blood or bone marrow are removed and grown in the laboratory. They are then exposed to a virus carrying the desired gene. The virus enters the cells, and the desired gene becomes part of the DNA of the cells. The cells are allowed to grow in the laboratory before being returned to the patient by injection into a vein.
  
• 2. In vivo, which means “inside the body” – No cells are removed from the patient’s body. Instead, vectors are used to deliver the desired gene to cells in the patient’s body.
  
• Currently, the use of gene therapy is limited. Somatic gene therapy is primarily at the experimental stage. Germline therapy is the subject of much discussion but it is not being actively investigated in larger animals and human beings.
  
• As of June 2001, more than 500 clinical gene-therapy trials involving about 3,500 patients have been identified worldwide. Around 78% of these are in the United States, with Europe having 18%. These trials focus on various types of cancer, although other multigenic diseases are being studied as well. Recently, two children born with severe combined immunodeficiency disorder (“SCID”) were reported to have been cured after being given genetically engineered cells.
  
• Gene therapy faces many obstacles before it can become a practical approach for treating disease.[14] At least four of these obstacles are as follows:
  

1.Gene delivery tools. Genes are inserted into the body using gene carriers called vectors. The most common vectors now are viruses, which have evolved a way of encapsulating and delivering their genes to human cells in a pathogenic manner. Scientists manipulate the genome of the virus by removing the disease-causing genes and inserting the therapeutic genes. However, while viruses are effective, they can introduce problems like toxicity, immune and inflammatory responses, and gene control and targeting issues

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2. Limited knowledge of the functions of genes. Scientists currently know the functions of only a few genes. Hence, gene therapy can address only some genes that cause a particular disease. Worse, it is not known exactly whether genes have more than one function, which creates uncertainty as to whether replacing such genes is indeed desirable.
   

3.Multigene disorders and effect of environment. Most genetic disorders involve more than one gene. Moreover, most diseases involve the interaction of several genes and the environment. For example, many people with cancer not only inherit the disease gene for the disorder, but may have also failed to inherit specific tumor suppressor genes. Diet, exercise, smoking and other environmental factors may have also contributed to their disease.

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2. High costs. Since gene therapy is relatively new and at an experimental stage, it is an expensive treatment to undertake. This explains why current studies are focused on illnesses commonly found in developed countries, where more people can afford to pay for treatment. It may take decades before developing countries can take advantage of this technology.
   
3. DNA Replication image from the Human Genome Project (HGP)
   
4. The Human Genome Project is an initiative of the U.S. Department of Energy (“DOE”) that aims to generate a high-quality reference sequence for the entire human genome and identify all the human genes.
   
5. The DOE and its predecessor agencies were assigned by the U.S. Congress to develop new energy resources and technologies and to pursue a deeper understanding of potential health and environmental risks posed by their production and use. In 1986, the DOE announced its Human Genome Initiative. Shortly thereafter, the DOE and National Institutes of Health developed a plan for a joint Human Genome Project (“HGP”), which officially began in 1990.
   
6. The HGP was originally planned to last 15 years. However, rapid technological advances and worldwide participation accelerated the completion date to 2003 (making it a 13 year project). Already it has enabled gene hunters to pinpoint genes associated with more than 30 disorders.[15]
   

CLONING

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• Cloning involves the removal of the nucleus from one cell and its placement in an unfertilized egg cell whose nucleus has either been deactivated or removed.
  
• There are two types of cloning:
  
• 1. Reproductive cloning. After a few divisions, the egg cell is placed into a uterus where it is allowed to develop into a fetus that is genetically identical to the donor of the original nucleus.
  
• 2. Therapeutic cloning.[16] The egg is placed into a Petri dish where it develops into embryonic stem cells, which have shown potentials for treating several ailments.[17]
  
• In February 1997, cloning became the focus of media attention when Ian Wilmut and his colleagues at the Roslin Institute announced the successful cloning of a sheep, named Dolly, from the mammary glands of an adult female. The cloning of Dolly made it apparent to many that the techniques used to produce her could someday be used to clone human beings.[18] This stirred a lot of controversy because of its ethical implications.
  

In Agriculture :

Improve yield from crops

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• Using the techniques of modern biotechnology, one or two genes may be transferred to a highly developed crop variety to impart a new character that would increase its yield (30). However, while increases in crop yield are the most obvious applications of modern biotechnology in agriculture, it is also the most difficult one. Current genetic engineering techniques work best for effects that are controlled by a single gene. Many of the genetic characteristics associated with yield (e.g., enhanced growth) are controlled by a large number of genes, each of which has a minimal effect on the overall yield (31). There is, therefore, much scientific work to be done in this area.
  
• Crops containing genes that will enable them to withstand biotic and abiotic stresses may be developed. For example, drought and excessively salty soil are two important limiting factors in crop productivity. Biotechnologists are studying plants that can cope with these extreme conditions in the hope of finding the genes that enable them to do so and eventually transferring these genes to the more desirable crops. One of the latest developments is the identification of a plant gene, At-DBF2, from thale cress, a tiny weed that is often used for plant research because it is very easy to grow and its genetic code is well mapped out. When this gene was inserted into tomato and tobacco see RNA interference cells, the cells were able to withstand environmental stresses like salt, drought, cold and heat, far more than ordinary cells. If these preliminary results prove successful in larger trials, then At-DBF2 genes can help in engineering crops that can better withstand harsh environments (32). Researchers have also created transgenic rice plants that are resistant to rice yellow mottle virus (RYMV). In Africa, this virus destroys majority of the rice crops and makes the surviving plants more susceptible to fungal infections (33).
  
• Proteins in foods may be modified to increase their nutritional qualities. Proteins in legumes and cereals may be transformed to provide the amino acids needed by human beings for a balanced diet (34). A good example is the work of Professors Ingo Potrykus and Peter Beyer on the so-called Goldenrice(discussed below).
  
• Modern biotechnology can be used to slow down the process of spoilage so that fruit can ripen longer on the plant and then be transported to the consumer with a still reasonable shelf life. This improves the taste, texture and appearance of the fruit. More importantly, it could expand the market for farmers in developing countries due to the reduction in spoilage.
  
• The first genetically modified food product was a tomato which was transformed to delay its ripening (35). Researchers in Indonesia, Malaysia, Thailand, Philippines and Vietnam are currently working on delayed-ripening papaya in collaboration with the University of Nottingham and Zeneca (36).
  
• Most of the current commercial applications of modern biotechnology in agriculture are on reducing the dependence of farmers on agrochemicals. For example, Bacillus thuringiensis (Bt) is a soil bacterium that produces a protein with insecticidal qualities. Traditionally, a fermentation process has been used to produce an insecticidal spray from these bacteria. In this form, the Bt toxin occurs as an inactive protoxin, which requires digestion by an insect to be effective. There are several Bt toxins and each one is specific to certain target insects. Crop plants have now been engineered to contain and express the genes for Bt toxin, which they produce in its active form. When a susceptible insect ingests the transgenic crop cultivar expressing the Bt protein, it stops feeding and soon thereafter dies as a result of the Bt toxin binding to its gut wall. Bt corn is now commercially available in a number of countries to control corn borer (a lepidopteran insect), which is otherwise controlled by spraying (a more difficult process).
  
• Crops have also been genetically engineered to acquire tolerance to broad-spectrum herbicide. The lack of cost-effective herbicides with broad-spectrum activity and no crop injury was a consistent limitation in crop weed management. Multiple applications of numerous herbicides were routinely used to control a wide range of weed species detrimental to agronomic crops. Weed management tended to rely on preemergence — that is, herbicide applications were sprayed in response to expected weed infestations rather than in response to actual weeds present. Mechanical cultivation and hand weeding were often necessary to control weeds not controlled by herbicide applications. The introduction of herbicide tolerant crops has the potential of reducing the number of herbicide active ingredients used for weed management, reducing the number of herbicide applications made during a season, and increasing yield due to improved weed management and less crop injury. Transgenic crops that express tolerance to glyphosate, glufosinate and bromoxynil have been developed. These herbicides can now be sprayed on transgenic crops without inflicting damage on the crops while killing nearby weeds (37).
  
• From 1996 to 2001, herbicide tolerance was the most dominant trait introduced to commercially available transgenic crops, followed by insect resistance. In 2001, herbicide tolerance deployed in soybean, corn and cotton accounted for 77% of the 626,000 square kilometres planted to transgenic crops; Bt crops accounted for 15%; and "stacked genes" for herbicide tolerance and insect resistance used in both cotton and corn accounted for 8% (38).
  
• Biotechnology is being applied for novel uses other than food. For example, oilseed can be modified to produce fatty acids for detergents, substitute fuels and petrochemicals.[citation needed] Potatos, tomatos, rice, tobacco, lettuce, safflowers, and other plants have been genetically-engineered to produce insulin[citation needed] and certain vaccines. If future clinical trials prove successful, the advantages of edible vaccines would be enormous, especially for developing countries. The transgenic plants may be grown locally and cheaply. Homegrown vaccines would also avoid logistical and economic problems posed by having to transport traditional preparations over long distances and keeping them cold while in transit. And since they are edible, they will not need syringes, which are not only an additional expense in the traditional vaccine preparations but also a source of infections if contaminated.[21] In the case of insulin grown in transgenic plants, it is well-established that the gastrointestinal system breaks the protein down therefore this could not currently be administered as an edible protein. However, it might be produced at significantly lower cost than insulin produced in costly, bioreactors. For example, Calgary, Canada-based SemBioSys Genetics, Inc. reports that its safflower-produced insulin will reduce unit costs by over 25% or more and reduce the capital costs associated with building a commercial-scale insulin manufacturing facility by approximately over $100 million compared to traditional biomanufacturing facilities[1].
  
• There is another side to the agricultural biotechnology issue however. It includes increased herbicide usage and resultant herbicide resistance, "super weeds," residues on and in food crops, genetic contamination of non-GM crops which hurt organic and conventional farmers, damage to wildlife from glyphosate, etc.[2][3]
  

CHEESE PRODUCTION :

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• Enzymes produced by micro-organisms provide an alternative to animal rennet – a cheese coagulant - and an alternative supply for cheese makers. This also eliminates possible public concerns with animal-derived material, although there is currently no plans to develop synthetic milk, thus making this argument less compelling. Enzymes offer an animal-friendly alternative to animal rennet. While providing comparable quality, they are theoretically also less expensive.
  
• About 85 million tons of wheat flour is used every year to bake bread[20]. By adding an enzyme called maltogenic amylase to the flour, bread stays fresher longer. Assuming that 10-15% of bread is thrown away, if it could just stay fresh another 5–7 days then 2 million tons of flour per year would be saved. That corresponds to 40% of the bread consumed in a country such as the USA. This means more bread becomes available with no increase in input. In combination with other enzymes, bread can also be made bigger, more appetizing and better in a range of ways.
  

BIOREMEDIATION

Bioremediation can be defined as any process that uses microorganisms, fungi, green plants or their enzymes to return the natural environment altered by contaminants to its original condition.

An example of a more general approach is the cleanup of oils spills by the addition of nitrate and/or sulfate fertilisers to facilitate the decomposition of crude oil by indigenous or exogenous bacteria.

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• Biotechnology is being used to engineer and adapt organisms especially microorganisms in an effort to find sustainable ways to clean up contaminated environments. The elimination of a wide range of pollutants and wastes from the environment is an absolute requirement to promote a sustainable development of our society with low environmental impact. Biological processes play a major role in the removal of contaminants and biotechnology is taking advantage of the astonishing catabolic versatility of microorganisms to degrade/convert such compounds. New methodological breakthroughs in sequencing, genomics, proteomics, bioinformatics and imaging are producing vast amounts of information. In the field of Environmental Microbiology, genome-based global studies open a new era providing unprecedented in silico views of metabolic and regulatory networks, as well as clues to the evolution of degradation pathways and to the molecular adaptation strategies to changing environmental conditions. Functional genomic and metagenomic approaches are increasing our understanding of the relative importance of different pathways and regulatory networks to carbon flux in particular environments and for particular compounds and they will certainly accelerate the development of bioremediation technologies and biotransformation processes.[22]
  
• Marine environments are especially vulnerable since oil spills of coastal regions and the open sea are poorly containable and mitigation is difficult. In addition to pollution through human activities, millions of tons of petroleum enter the marine environment every year from natural seepages. Despite its toxicity, a considerable fraction of petroleum oil entering marine systems is eliminated by the hydrocarbon-degrading activities of microbial communities, in particular by a remarkable recently discovered group of specialists, the so-called hydrocarbonoclastic bacteria (HCB).[23]
  

Main Types of Bioremediation :

Biostimulation -- Nutrients and oxygen - in a liquid or gas form - are added to contaminated water or soil to encourage the growth and activity of bacteria already existing in the soil or water. The disappearance of contaminants is monitored to ensure that remediation occurs.

Bioaugmentation -- Microorganisms that can clean up a particular contaminant are added to the contaminated soil or water.

Bioaugmentation is more commonly and successfully used on contaminants removed from the original site, such as in municipal wastewater treatment facilities.

To date, this method has not been very successful when done at the site of the contamination because it is difficult to control site conditions for the optimal growth of the microorganisms added.

Intrinsic Bioremediation -- Also known as natural attenuation, this type of bioremediation occurs naturally in contaminated soil or water.

This natural bioremediation is the work of microorganisms and is seen in petroleum contamination sites, such as old gas stations with leaky underground oil tanks.

oBioremediation technologies can be classified as in situ or ex situ.

In situ bioremediation involves treating the contaminated material at the site

Ex situ involves the removal of the contaminated material to be treated elsewhere

oSome examples of bioremediation technologies are bioventing, land farming, bioreactor, composting, bioaugmentation and biostimulation.

oIn situ-Bioremediation that can be used at the site of contamination.

oEx situ- On contamination removed from the original site.

oThere are a number of advantages to bioremediation, which may be employed in areas which cannot be reached easily without excavation (penggalian).

E.g:hydrocarbon spills (or more specific: gasoline) may contaminate groundwater well below the surface of the ground; injecting the right organisms, in conjunction with oxygen-forming compounds, may significantly reduce concentrations after a period of time