Genetically Modified Organisms - Background

What is genetic modification   -^-

Prior history   -^-

Johann Mendel was born in Heinzendorf, Austria (now Hyncice, Czech Republic), and choose the name Gregor when he entered the Augustinian Abbey of St Thomas in Brno in 1843. Gregor Mendel is often called the father of genetics since he was the first to understand the basic principles by which traits (genes) are transmitted from parents to offspring, when experimenting with peas. His ideas and hypotheses were long neglected and even Darwin had only an obscure idea of how such information was passed on from from the parent body to pass on to the offspring. It was around the year 1900 when scientists rediscovered Mendel's laws on the genetic linkage of traits. In the following decades the molecular nature of genes, their cellular location and their products were discovered, which led to a revolution in biology that is still gaining momentum.

For many centuries, and all over the world, people have been selecting individual plants and animals with particular traits as starting material for breeding purposes. Experience told them that crossing these parents could result in offspring with favourable traits from both parents.

It is remarkable what has been accomplished with this technique. Think alone of the numerous dog breeds that now exist, which all are descendents from only a few original wild races of dogs. Other examples are the current agricultural crops, husbandry animals, or the availability of large varieties of cut flowers.

Although these organisms were all shaped to the needs of mankind, they were not all the result of a methodical selection that was oriented toward a predetermined standard. Unconscious selection, or automatic selection as it is sometimes called, could have been responsible for most of the differences that distinguish domesticated seed crops from their wild progenitors, including traits like increased seed size, loss of natural dispersal mechanisms, even and rapid seed germination and simultaneous ripening. Unconscious selection, more common in ancient times, may be defined as non-intentional human selection and was only driven by the way the organism was used by humans.

In contrast, methodical selective breeding requires great care and knowledge. It is capable of rapid change in specific traits, such as the quantity and quality of milk production or resistance to diseases in agricultural crops. Selective breeding aims to tailor the plant or animal for a certain application and thus can be seen as a process of evolution by human selection. Reaching an exact and desired combination of favourable traits from starting breeding materials is not easy. In reality, the progeny will have a mix of traits, both wanted and unwanted. It will take a number of breeding cycles to eliminate negative traits and build on the positive ones.

Both the automated and targeted selections are part of a traditional breeding approach. They rely on the available genetic resources of an organism by getting rid of unwanted traits while retaining the desirable genes when further crosses are made. Resistances to certain diseases, or the capacity to produce certain substances (i.e. pharmaceuticals) that are not in the gene pool of a certain organism can therefore not be introduced by making use of traditional breeding techniques.

Moving around genes   -^-

All living organisms have DNA (deoxyribonucleic acid) in their cells, often in a confined structure that is called the nucleus. The DNA molecules function as a library, it is the organism’s genetic blueprint. DNA molecules can be very long containing millions of repetitive elements, called nucleotides (guanine, adenine, thymine and cytosine). It is the exact order of these nucleotides that forms instructions for making the materials (proteins and RNA molecules) which an organism needs to function. The DNA sequence also includes the information that is involved in regulating the use of this genetic information. The segments of DNA that are associated with the synthesis of real cellular components are called genes. Other parts of the DNA may be used to shape it into folded structures (chromosome) or have a function in determining when genes must be active.

The instructions in genes allow living cells to make proteins. Proteins can be used as structural parts of the organism, they can have a role in regulating cellular processes or they may be involved in catalysing chemical reactions. In the latter case the protein is called an enzyme. It is clear therefore that a single gene cannot code for a wing or a complete structure such as an eye. However, there are genes that have a crucial role in the initial steps when eye formation starts or how the shape of the wing will look like. Most often, however, multiple genes are jointly responsible for a specific property that could be the target of a breeding project. The more genes are involved in a targeted trait, the more complicated the selection process may be.

The terms "genetic modification", "genetic manipulation" (GM), "genetic engineering", "recombinant DNA technology", "gene cloning" and "gene splicing" all describe the process of identifying, isolating, changing and/or (re)introducing genes in an organism, usually in a laboratory setting. Genetic modification is a modern biotechnology technique developed in the past 30 years allowing scientists to artificially change an organism's genetic code. Since living organisms conscientiously duplicate their genetic material, any foreign or modified stretch of DNA in a so-called transgenic or genetically modified organism (GMO) will be passed on to its progeny. Usually, a small section of DNA from one organism is introduced into the DNA of another with which it would not normally interbreed. The technique owes to the fact that all organisms have a huge number of genes in common. Therefore DNA information from a given organism may very well be suited to function properly in another unrelated organism from a different species. Changing or introducing a few select genes may introduce the capability of making new substances or provide an organism with new or different capabilities.

An example is the synthesis of human insulin through the use of genetically modified bacteria, which is used as a medication by diabetes patients. The following steps were taken to accomplish the routine production of this medication:

- DNA is isolated from human cells and the long DNA molecules are broken into small pieces.
- These DNA fragments are introduced into laboratory bacteria that can easily be cultured in large quantities.
- Only a very few bacteria would have received the fragment of DNA that contains information for the production of the human insulin hormone protein. Clever techniques allow the identification and isolating of only those bacteria from the vast majority of others.
- A human gene is not right away recognised in bacteria, so no insulin will be produced. Therefore the stretch of human DNA, that is faithfully copied in these bacteria, is isolated once again.
- The sequence of nucleotides of the isolated fragments of DNA can be analysed and modified in such a way that the bacteria will recognise it and synthesize the insulin protein.
- Upon reintroduction of this modified DNA in a new batch of bacteria, individual bacteria can be isolated that demonstrate the desired production of insulin.
- Insulin consists of two separate proteins, thus in reality this process was carried out for both proteins (two genes, two separate stretches of human DNA), which needed to be combined and linked.

The above described steps only became possible because molecular biologists discovered enzymes which can cut and join strands of DNA. In figure 2 it can be seen that a restriction enzyme is used to open the DNA strand and DNA ligase would close it again. If a fragment with the human gene for insulin is present, it can be included in the circular piece of DNA.

For the introduction of the modified pieces of DNA into target organisms so called vectors are used. These are self replicating molecules of DNA such as plasmids for bacteria or viruses that are able to infect eukaryotic cells. Other methods include brute force physical methods like bombarding cells with DNA coated micro-particles or stressing cells in an electrical field so pores are being created through which DNA can slip into the cells.

Genetic modification does not necessarily mean that a gene from another organism is used to create a GMO. The technique can also be used to extract, modify and reintroduce the gene(s) in the same organism. It can also be useful to turn down gene activity (silencing), such as in oilseed crops where this is done to prevent the production of unhealthy oils. Recombinant and molecular DNA technologies have even made possible to build customized stretches of DNA and thus synthesise genes with altered properties.

Surprisingly, genetic modification is not only restricted to human activity. Different species of the soil bacterium Agrobacterium are capable to transfer a specific piece of their own DNA to certain plant cells, and put them to work in resulting tumour-like tissue for their own benefit. Although this is an example with a limited effect, it can be argued that GM is also a natural phenomenon. The property of these bacteria has subsequently been exploited as a tool when introducing for example human DNA sequences into plant cells.

Genetically modified microorganisms   -^-

Microorganisms are a very diverse group of independently living organisms that are found from the coldest to the hottest places in the world, in the air, on land and in water environments. The group includes bacteria, fungi, algae and viruses. Most consist of just one cell and not visible to the naked eye. But sometimes the cells may combine into structures, such as is true for fungi and algae, or the single cell is huge so it is visible to the human eye.

Some microorganisms have a long history of usage in traditional food (e.g. cheese) and beverage (e.g. beer) preparation, but many others are pathogenic since they invade and grow within other organisms, causing diseases among people, animals or plants. Bacteria were the first microorganisms to be modified genetically by altering their own genes, or by introducing genes from other organisms. Bacterial systems lend themselves very well to genetic manipulation in part because of the ease of handling in the laboratory and their rapid reproduction rates. The model bacterium Escherichia coli has served as the model bacterium and the workhorse in laboratories for several decades now. It is now routinely used for replicating and altering genes that are subsequently introduced into plants or animals. For many other organisms the genetic makeup is now also fully known, and they can also be genetically altered for either scientific, medical or commercial purposes.

A microorganism can be an excellent choice for the production of specialty products or to carry out a specific task in a process, because of their great variety of natural properties and the wide range of environmental adaptations and growth properties. Bacteria are now routinely used to produce non-bacterial proteins such as industrial enzymes and a variety of pharmaceuticals. Genetically modified microorganisms (with adjusted properties) can also be put to work in biodegradation, bioremediation, crop protection or a range of other, sometimes exotic, applications.

Industrial enzymes   -^-

The most common application of genetically modified microorganisms is in the production of industrial enzymes. The isolation of such proteins from the fermentation fluid is the simplest way of harvesting, therefore often bacteria like Bacillus or fungi like Aspergillus are used, having excellent capacities to produce and secrete enzymes. Examples of industrial enzymes are: lipases, subtillisin (washing powders), amylases, pectinase (food industry), cellulose, protease (leather, textile industry).

Pharmaceuticals   -^-

Before it was possible to create GM microorganisms that would produce specialty pharmaceuticals, some of these could only be isolated from animal sources, human fluid donations or even corpses. They were in limited supply, expensive and the drugs were either not fully human compatible, or their purity was not optimal. Biotechnology now offers a cheap and efficient way of producing high quality drugs using recombinant DNA technology. Typically, these drugs are characterized by a high and specific activity combined with an optimal safety record. They include hormones, enzymes, growth and blood coagulation factors, antibiotics and vaccines. The biochemical machinery of the genetically modified organism of choice will translate and produce the desired protein(s) when cultured in large bioreactors, from which the product is isolated and purified.

Bioremediation   -^-

Applications for genetically modified microorganisms in bioremediation have received a great deal of attention, but have so far largely been confined to the laboratory environment. The main reasons for this are that laboratory bacterial strains were often out competed by bacterial species that are present naturally, that indigenous populations of bacteria were able to do the job as well when stimulated for growth or that regulatory risk assessment concerns prevent the release of the recombinant organisms.

Still many types of toxic wastes are not degraded naturally because of a lack of organisms that are able to metabolise the compounds. Thousands of man-made chemicals do not exists naturally, these are called xenobiotics, and no organisms have yet evolved that will be able to cope with them.

One of the areas, where genetically modified organisms are likely to be used include the biodegradation of polyaromatic hydrocarbons (PAHs), whose occurrence in the soils is due to spills or leakage of fossil fuels or petroleum products. Bioremediation also promises to offer solutions when soils contain too high levels of certain heavy metal salts. Genetically modified bacteria have been created that are able to transform the salt into the metallic form, so it will not leak to underground waterways.

Biodegradation   -^-

An attractive alternative for the current, oil-based, fuels is bio-ethanol - alcohol produced from agricultural crops. Thus far commodities of high value such as sugar or starch are used for this process, but agricultural leftover by-products also have high caloric value. Unfortunately these materials are mainly in the form of cellulose and lignin, which cannot be used by bakers yeast that would produce the end product ethanol. Several research activities show promising results when either the yeast strains are provided with genetic information coding for the proper degrading enzymes, or when other GM microorganisms are created that pre-process the waste.

Similar initiatives concentrate on the conversion of bio-waste into methane. The anaerobic decomposition of livestock and poultry manure, common to manure heaps and slurry tanks, leads to large amounts of methane production due to its large organic carbon content. Similarly, the processing of industrial and domestic waste water and sewage can also produce significant amounts of methane. Since this is a natural process, genetically modified organisms could only offer a contribution if special conditions need to be met, or the composition of the waste poses problems.

Crop protection   -^-

Microorganisms have also been modified through the techniques of genetic engineering to protect crops or aid their growth. The most prominent example is the natural occurring bacterium Bacillus thurengiensis. The soil bacterium happens to synthesise a protein (Bt) that will kill the larvae of a certain group of insects that otherwise would damage the crop. The gene for the trait has been transferred to plants as well as to other bacteria with the option to generate an improved tool for crop protection.

Among other proposed strategies and examples of this class of genetically modified microorganisms are:

- bacteria that were supplied with a gene for the enzyme chitinase, that would suppress fungal growth in the soil.
- production of a-virulant pathogenic bacteria that would out compete and protect crops against the naturally occurring bacteria that cause a plant disease. - antibiotic production by microorganisms that live in close proximity of the plant roots, causing suppression of the growth of pathogenic bacteria.
- provide plant associated microorganisms with genes that have a function in liberating nutrients from soil particles (such as iron).

Surprising ideas   -^-

- Researchers at Tel Aviv University have coated a microchip with genetically modified bacteria that can be used to measure water quality. The bacteria will light up when they come in contact with pre-determined pollutants and communicate with monitoring systems.
- Researchers at the University of Nebraska have provided the bacterium Cupriavidus metallidurans, that converts ionic gold into metallic gold, with a gene that will cause light emission when the bacterium finds the gold ions.
- Missouri Western State University researchers reported to have created ‘bacterial computers’ with the potential to solve a classic mathematical problem known as the Hamiltonian Path Problem.
- The bacterium Streptococcus mutans causes tooth decay when it consumes leftover sugars in the mouth and produces lactic acid that corrodes tooth enamel and ultimately causes cavities. Researchers modified the bacterium so it would not be able to produce lactic acid anymore. When these bacteria gain predominance in a person's mouth, this would reduce the formation of cavities.

Safety   -^-

The vast majority of experiments with and applications of genetically modified microorganisms is inherently safe, since they will not leave the research or production facility. Even if they would , then most laboratory strains would not survive because of their nutritional requirements. Safety is thus built into the experimental design. Nevertheless regulation is in place to review the conditions of facilities, production methods and waste removal.

However, if GM organisms are introduced in the environment on purpose, for a tested task, risk assessments must be submitted to the regulatory authorities for approval before such work can commence. Decisions are taken on a precautionary basis to allow some margin of safety, especially where there is any uncertainty over the risks.

Genetically modified plants   -^-

The genetic modification of plants has been undertaken in the last decades with different objectives:

- production of improved agricultural crops with better agronomic properties like disease resistance, growth characteristics, ripening time or yield.
- improvement of the quality of the crop's product such as starch composition, quality, presence of health improving compounds (vitamins) or storage properties.
- plants can be used for bioremediation and take up pollutants from the atmosphere or from the soil. Certain plants can actively take up heavy metals from the soil and thus play a role in environmental protection.
- plants can, similarly to bacteria, be used as hosts for the production of pharmaceuticals. It would depend on the product, the origin of the gene construct, its use whether plants are a good choice. It has been suggested for instance that tomatoes, bananas or potatoes could be genetically modified to produce edible vaccines for infectious diseases including cholera, hepatitis B and infectious diarrhoea.

The first generation of GM crops   -^-

The benefit of the first generation of genetically modified plants, as seen through the eyes of the consumer, was not very obvious. The traits that were introduced into crop plants were selected for their agronomic properties and would primarily benefit farmers, industry and possibly the environment:

- Herbicide tolerance traits allows farmers to spray herbicides for the removal of weeds while the crop is developing. A clear advantage is that spraying can be done only when needed and the need for ploughing is reduced, resulting in less soil erosion. The risks associated with the use of herbicide tolerant crops are: emergence of tolerance to herbicides in weeds, misapplication, herbicide drift, and the need for timely application. However, similar risks are associated with conventional weed management systems, where herbicides are used as well.
- Damage by pests is an important and diverse problem in agriculture because of the wide range of pests and the different mechanisms of infection. A range of crop protection technologies have developed and most rely on the use of chemicals. The development of pest-resistant crops is therefore a major objective towards achieving sustainable agricultural practices. Genetic modification technologies allow the introduction of genes that for instance provide specific resistance against plant viruses, pathogenic bacteria or moulds. Other genes can be used that repel or are toxic for insects.

A widely used trait that is effective against insect damage originates from a bacterium Bacillus thuringiensis. The now called Bt protein is toxic for a class of insect larvae, since it causes the destruction of cells in the lining of their gut. After introduction and expression of this gene in plant cells, full resistance was reached against insect voracity. Examples are the Colorado potato beetle that completely can defoliate a potato crop and the European corn borer caterpillars that damages the ears and stalks of maize. Even secondary infections by molds, that take advantage of the damage caused by insects in maize, were reduced. The Bt protein does not necessarily need to be expressed in plant cells, the intact bacterium can also be sprayed on the crops, as is indeed done in plots that use organic agricultural methods.

Present and future generations of GM crops   -^-

New generations of GM crops have enhanced agronomic and nutritional properties as well as traits to improve their use in industrial processes. The development of crops with such improved yield characteristics and of GM foods with enriched nutrients, with improved functionality, and health promoting activities will all be of direct benefit to the consumer. To reach such goals it is necessary to bring about more complicated changes in the plant metabolic pathways, thus modifications involving more than just one gene.

Today there are many examples of such advanced genetically crops, although most are not yet commercially available:

- potatoes or maize with altered starch and protein composition allow the development of crop varieties with superior taste, better structure and improved nutritional value. Also special non-food varieties have been selected that are used in industrial starch production with improved energy efficiencies.
- some people suffer from allergens that can be present in natural foods, such as in nuts or fruits. Genetic modification can, with sufficient knowledge about the metabolic pathways in plant cells, modify the crop so these allergens will not be made.
- foods can spoil readily and sometimes crops need to be harvested in unripe state (bananas) which does not improve the quality of the end product. Knowledge about the processes that cause spoilage after ripening can extend the shelf life of the products and increase their nutritional value over an extended period of time.
- an early promise has been the generation of edible vaccines for infectious diseases like cholera, hepatitis B, and diarrhoea, when produced by genetically modified plants and administered as a food component. If successful such plants could be of great help to fight diseases and offer a cost effective method of producing them.
- bulk production of oils or carbohydrates can serve as alternative sources of energy, especially if crops are adapted to produce only oils of desired type's, or the compounds can be harvested easily.

An exceptional example of the clever nutritional modification of rice has been the development of so called golden rice. The Swiss researchers Ingo Potrykus and Peter Beyer undertook the task of introducing the metabolic pathway leading to the synthesis of beta-carotene, a precursor of pro-vitamin A, in the rice endosperm which is the part of the seed that is eaten. From 1992 until 2000, they and their collegues worked to accomplish the introduction of four new genes into rice and thus engineered an entire biosynthetic pathway. Golden rice, so called because the seed obtained a yellowish colour, was developed as a fortified food to be used in areas where there is a shortage of dietary vitamin A. People with Vitamin A deficiency suffer from irreversible blindness and it may lead to death. It is a great problem in many countries in Africa and South East Asia where UNICEF runs vitamin A supplementation programs for children under 5. The introduction of golden rice varieties would expand and simplify the current effort.

While there is a wide range of options and potentials for the use of genetically modified crops, there are a number of reasons for the slow adoption of the technology. A variety of products (industrial bulk and pharmaceutical specialties) can efficiently be made with microorganisms which requires less complicated provisions, since these are kept indoors. Also, GM food products have met with significant opposition from environmental and anti-globalization activists, causing breeding companies and governments to slow down their development.

Also, the future coexistence of genetically modified crops with conventional and organic crops has raised significant concern in many European countries. In the fields measures must be in place to make sure that crops will not mix when sown, nor when harvested. During the growth period there may also be possibilities for cross-pollination, therefore GM pollen may fertilise a organically grown crop that is located nearby. For crops like potatoes this is not an issue, since the root tubers are harvested and not the seed, but for cereals and oilseed rape proper distances must be kept between the fields. Since there is separate legislation for GM crops and a high demand from consumers for the freedom of choice between GM and non-GM foods, measures would also be required to separate foods and feed produced from GM plants from conventional or organic foods.

Food and gene technology   -^-

Science has moved at such a rapid pace that consumers have been taken by surprise and the safety of genetically modified foods has become a concern. Humanity has a long tradition with conventional improvements of crops and livestock through breeding programmes and is familiar with their products over a long period of time. While quite poisonous potatoes can be bred, there is high confidence that nothing spectacular can go wrong in conventional breeding programmes. These issues are perceived differently for the use of genetically modified organisms and the products thereof. Concerns about GM foods fall into the following categories: environmental hazards, human health risks, economic concerns and ethical considerations.

Health concerns   -^-

The human health risks that are thought to be relevant considering are the following:

Toxins: it is perfectly possible to generate GM plants that produce toxic substances. Such plants also exist in nature and the potato is a good example. All potatoes contain natural toxins called glycoalkaloids. The levels are usually low but higher levels are found in potato sprouts, and the peel of potatoes that taste bitter. The toxins are produced by the plant in response to stress such as micro-organisms, UV light, and damage such as bruising. The amount of toxin depends on the type of potato and the growing conditions. Potato breeders make sure that levels stay low in varieties that are used for consumption. Likewise breeders who generate genetically modified crops make sure that no toxins are present. Genetic modification could even be used to remove known toxins from food plants.

Examples of toxins in natural foods are: Seeds of apple, pear, apricot and peach contain a substance called amygdalin from which hydrogen cyanide can be produced in the stomach. Parsnips commonly contain a group of natural toxins known as furocoumarins. Rhubarb contains oxalic acid that can cause cramps, decreased breathing and heart action, convulsions and coma (probably after eating a field of rhubarb).

Allergenicity: about 1 - 2% of adults and about 5% of children display food allergies. Around 90% of food allergies are induced by peanuts, soybeans, vegetables, fruits, milk, eggs, cereals, nuts, some fish and shellfish. The allergic reaction is generally caused by a single component, mostly a protein, called allergen. The compound causes an allergic reaction when the food item comes into close contact with our blood system, principally in the intestinal mucous membranes. The body mistakenly takes the food component as an invading parasite and starts a defence reaction. In theory it is possible that genetically modified crops contain such an allergen. The same technique can however be put to work to remove the compound from crops known to cause allergies.

Nutritional effects: it has been put forward that there could be unintended effects caused by the technique of genetic modification or the nature of gene insertion. The nutritional value of the resulting crop could then for instance be reduced.

Legally enforced assessments have been set up for the rigorous evaluation of GM organisms and GM foods relative to both human health and the environment. GM foods currently available on the international market have passed risk assessments and have not shown to present risks for human health. In addition, no effects on human health have been shown as a result of the consumption of commercialised GM foods by the general population in the countries where they have been approved.

Novel varieties that have been bred by conventional methods or traditional foods are not evaluated through such a strict regime because it is the general believe that traditional foods are inherently safe. Little information is available on the differences of nutritional values between conventional varieties of food plants.

Stability of the inserted gene: since genes have been put into plants, it is thought that they could also move out and taken up by bacteria or even human cells in the gastrointestinal tract. Although transfer of genetic materials has been shown to be very infrequent events and thus is unlikely to occur, it has been accepted that antibiotic resistance genes, which are helpful in creating GMOs, should either be avoided or removed.

Labelling: it is impossible to see, taste or smell whether foods contain materials that were produced with the use of genetically modified organisms. Consumer responses and public attitudes are the factors that will define the role of genetic modification in our society in the long run. Labelling of foods is a clear method to offer consumers a choice and this route has been taken in Europan countries, amongst others. Arguments not to label GM foods in the U.S. include: i) GM foods are substantially identical to non-GM foods, therefore there is no need to label. ii) Labels are likely to mislead consumers by implying a warning, iii) It is economically not feasible to separate GM crops from non-GM crops, either on the field, harvested materials or the products thereof.

Environmental concerns   -^-

The variety of traits that confer tolerance to herbicides are quite limited, therefore increased use of such GM crops results in the wide scale use of only a small class of herbicides. This situation may lead to the following problems:

- increased use of a limited number of herbicides.
- weeds with some tolerance have an advantage, leading to a reduction in the spectrum of other plants, thus loss of biodiversity.
- the herbicide trait may outcross to conventional crops or related species in the wild, giving a boost to the previous issue.

Many crops have been modified to carry a Bt gene, that confers resistance against damage caused by larvae of a class of insects called Lepidoptera. (see section "The first generation of GM crops"). Again, because of the global usage of such GM crops, resistance is thought to emerge, rendering the use of this tool useless. Organic farmers fear this sequence of events, because they use the intact bacterium as a spray to prevent damage by these insects. Other problems are the secondary effects of the Bt proteins in the crop plants on non-target insects. The Bt protein in pollen, roots and left-over materials after harvest may influence the natural insect ecosystem.

Traditional crops have been bred in the past to grow in different climates and regions of the world. Therefore the number of utilised varieties (genotypes), of a specific crop like wheat, maize or rice is large. This is a good situation, since plant diseases will not spread quickly when different varieties have different susceptibilities.

With the creation of successful genetically modified crops it is likely that the diversity in crop varieties will decrease because of the cost of producing a single new GM variety. Moreover, the variety of traits that are introduced, such as pest resistance (insects, plant viruses), are even more limited. If, therefore, relative uniform fields of crops with a certain pest resistance are grown globally, there is an increased probability that tolerance will emerge and the pest will spread more quickly than ever.

The environmental safety aspects of the use of GM crops may vary considerably according to local conditions. The change in agricultural practices may lead to a decreased use of the important practice of crop rotation that also influences the (soil) ecosystem. It must however be noted that the introduction of pest resistance genes into crop plants greatly reduces the need for chemical spraying. While this benefit would sound like a winner for organic agriculture, it is not, since GM plants and their products are not considered organic.

Genetically modified animals   -^-

Animal breeders have succeeded in creating today's high quality livestock through careful selection and breeding. While this powerful tool will long serve to improve the health and production capacity of cattle, fowls and other animals used in the human food chain, Novel genetic technologies hold promise of new approaches. The cloning of animals by nuclear transfer and genetic modification of their traits are two such techniques, but what would be the purpose of using these techniques?

Genetic modification   -^-

- Livestock could, in theory, be genetically altered to raise milk or wool production or enhance the quality of their meat, while lowering costs to farmers. So far no transgenic animals, to be used for food products, have been approved anywhere in the world. Some people object to the technology because it is unethical to view animals as production machines for food. Another disadvantage is a possible reduction of genetic diversity if only a few successful individuals are further propagated naturally or even by cloning.
- Genetically modified animals can also be essential to produce human proteins that are in short supply and needed for the treatment of diseases. Cattle, sheep, goats, chickens, rabbits and pigs have been genetically modified with the aim of producing human proteins that are useful, often in milk and generally as medicines. The introduction of genes at specific location in the genome and their expression in proper tissues is typically difficult to achieve and many initiatives have been shelved. Recently however, the U.S. Food and Drug Administration has approved the first ever biological product produced by genetically modified goats. ATryn is an anticoagulant, used for the prevention of blood clots. It is for patients who have a rare disease known as AT (hereditary antithrombin) deficiency.
- The genetic modification of animals, predominantly of mice, is widely used for scientific research. Transgenic animals can be used to help understand and develop treatments for both human and animal diseases. Mice are easy to keep, they grow fast and are sufficiently similar to humans to study the roles of single genes. For this purpose, so called knockout mice have been produced that have a particular gene or set of genes inactivated for research purposes.
- In 2003, a tropical zebrafish (Danio rerio) was given a gene from a jellyfish that causes it to glow in the dark, and so did the fish. Commercialisation under the name 'Glofish' was only approved in the U.S., with the exception of California, since the fish would not propagate in the wild, nor used as food nor breed with other species. In the same category of pets would fit the initiative by the 'Transgenic Pets' company (Syracuse, NY, U.S.) to produce allergy-free cats. Funding problems ended the project.
- Transgenic fish, like salmon and carp, have been equipped with multiple or enhanced genes that code for growth hormone. The fish grow faster and larger, while using less feed. The "blue revolution" - like the green revolution in biotech agriculture - has not started yet because of consumer worries and because the fish will surely escape in the wild and then breed with, or even overtake natural populations. Nevertheless, there are potentials for transgenic fish to reduce environmental impacts of current aquaculture methods and to make healthier food (increased omega-3 fatty acid levels in fish).
- In 2009 scientists in Japan announced the genetic modification of the first primate species, marmosets (Callithrix jacchus, a small monkey species). The achievement marks the successfully introduction of a foreign gene and expression of the green fluorescent jellyfish protein in all the cells of a primate. Potentially the transgenic marmosets can be used to study diseases and disorders that affect higher brain structures and could serve as a model for human neurodegenerative diseases.

In most countries technologies for the direct genetic modification of animals are strictly controlled. The EU legislation governing the 'contained use' and 'deliberate release' of genetically modified organisms includes animals. This legislation is designed to protect both human health and the environment.

Reproductive cloning   -^-

Cloning to create identical individuals, or asexual reproduction, means that an individual is created without the involvement of any intact egg and sperm cells. It is a technology used to generate an animal that has the same nuclear DNA as another animal. The asexual reproduction of for instance farm animals potentially offers possibilities to generate lots of quality cattle of a successful type. The technology could also be used for humans, but that is not likely to happen because of ethical issues.

The simplest way of cloning is the artificial generation of twins or multiple births by separating cells from very early embryonic stages and transferring them independently to the uterus of female hosts where they will then continue to develop until birth. In cows, such embryonic clones have a slightly extended gestation period, increased birth weight and mortality.

Scottish scientists at Roslin Institute created sheep "Dolly" as the first cloned animal from an adult individual. This made her the identical twin of a sheep that was six years older! The technique (somatic cell nuclear transfer, SCNT) involved the introduction of the genetic material of an adult donor cell (the single genetic "parent" of Dolly) into an egg cell from which the nucleus had been removed. The resulting artificial embryo then developed into a sheep with genetic properties that were (almost) equal to the original adult donor cell. The news immediately aroused worldwide interest and concern because of its scientific and ethical implications.

Before demonstration of the feasibility of this approach, scientists believed that the genetic material of an adult differentiated cell was permanently changed and specialised to only express genes that were relevant for the specific cell type. However, the fact that Dolly's grew up normally proved that the genetic material of specialised adult cells, in Dolly's case from an adult sheep's udder, could be reprogrammed to give rise to embryonic totipotent stem cells.

A SCNT clone is not an absolute identical clone of the donor animal. The egg cell contributes some DNA sequences that are located in mitochondria, which are cellular organelles that generate most of the cell's chemical energy. Only the clone's chromosomal or nuclear DNA is the same as the donor.

Also, the SCNT technique is not without problems. Only 0.1-1.0% of all eggs that receive transplanted cell nuclei from adult mammals result in the birth of a live animal. Dolly was the 300th try and many treated egg cells never developed or gave rise to deformed and stillborn animals. It is probable that the reprogramming process of the donor DNA in the recipient egg cell often fails, resulting in high rates of incapable embryo's.

Reproductive cloning is thus expensive and highly inefficient. In addition, cloned animals live in poor health and die early, probably because of a range of defects. These include compromised immune function, growth disorders, higher incidence of tumour development and others. Scientists have discovered that about 4% of the genes in the liver cells of cloned mice function abnormally. The abnormalities do not arise from mutations in the genes but from changes in the normal activation or expression of certain genes. The reprogramming of the donor DNA may have failed partially, or the requirements for a process called "imprinting" are not followed. Normally, in cells two copies are present, one from the father and one from the mother, and both can be active. However for some genes only one copy is active and the other must thus be marked to keep silent. Defects in the genetic imprint of DNA from a single donor cell may also lead to some of the developmental abnormalities of cloned embryos.

The genetic modification of humans   -^-

Reproductive cloning   -^-

Therapeutic cloning, also called embryo cloning or biomedical cloning, is the procedure to harvest stem cells that can be used to study human development and to treat disease. Stem cells are pluripotent, which means that they can be used to generate virtually any type of specialized cell in the human body. This property is important for the understanding of how cells develop from foetus to adult and for the development of cell therapies.

Stem cells could thus, at least in theory, be used to treat diseases in any body organs or tissues by replacing damaged and dysfunctional cells. If the stem cells originate from the patient itself, then the cells, the cultured tissue or organ would have the sick person's original DNA. It would mean that there is no danger of rejection and there would be no need to take immunosuppressant drugs. This technique would be vastly superior to relying on current organ transplants from other people.

It may seem far fetched that reprogrammed stem cells to become nerve or brain cells could cure Parkinson's or Alzheimer's disease. However current research already shows promising results with stem cells, that were harvested from the bone marrow of patients with heart failure, replacing damaged blood vessel tissue in the heart of the same patients. (see EUSEM film "Beat of the Heart"). Another interesting possibility is to generate tissue cells from individuals with genetic defects and test in laboratory dishes (in vitro) whether chemicals or biological substances could correct the faulty behaviour of the cells.

It should be noted that there is a long way to go. Much more research is needed to obtain more knowledge about the nature of differentiation factors that determine what type of cell a given stem cell may become. The next step will be to obtain sufficient tissue of proper quality. Artificial skin, cartilage and blood vessels can already be cultured from stem cells and are cartilage cells have even been grown in the shape of an ear or nose. In the distant future lies the production of full organs. It is thought that stem cells could differentiate and grow on moulds to reach certain shapes, but organs are often built from many different cell types and have a relatively complicated three-dimensional structure. For instance, in order to make a kidney it would be necessary to arrange all the different cell types correctly in relation to one another for the organ to be able to perform its function.

How are stem cells obtained?   -^-

During the initial phases when a fertilised egg cell starts dividing and forms an embryo, all cells are still identical. They can develop into all types of future body cells as well as the foetal membranes, placenta, umbilical cord. These cells are therefore called totipotent stem cells. As soon as the foetus gets a distinctive shape (the blastocyst stage), the inner cells differentiate into pluripotent embryonic stem cells, which still can form all of the body cells but not the foetal membranes, placenta, umbilical cord.

The embryonic stem cells can make any one of the 220 different cells in the human body and they can reproduce themselves many times over. These are the stem cells of choice for therapeutic cloning activities and they can be obtained from surplus in vitro fertilized (IVF) eggs. While it is certainly possible to produce early embryos in test tubes for research only, this is ethically not accepted. However, each year, hundreds of thousands of poor-quality embryos are discarded during the course of IVF, and these could provide an ethically acceptable source of stem cells for research.

Aborted foetuses also carry pluripotent stem cells in their genital tissue, which can be isolated. However, these cells could not be cultured with the same success as IVF derived cells in the laboratory.

Stem cells can also be harvested from the umbilical cord blood, which remains in the cord and the placenta after the baby is born. Most cord blood stem cells are hematopoietic cells that only differentiate into different types of blood cells, such as red blood cells, white blood cells and platelets. Since this is still a valuable source of cells when treating blood related diseases such as leukaemia etc., initiatives have been taken to collect and store the material in cord blood banks for possible future use. It is not yet clear what curative potential of these stem cells will be. Commercial firms have nonetheless made unsubstantiated claims promising insurance of infants or family members against serious illnesses in the future.

Adults also have stem cells present in the bone marrow, muscle, brain, lungs and other organs and tissues. Adult stem cells, also called somatic stem cells, may remain non-dividing for long periods of time. But when the need arises to maintain or repair the tissue, cell division is activated. Scientists have thought that adult stem cells would only be capable of giving rise to the same type of tissue from which they originated. Recent research has shown, however, that the cells may be induced to behave more like the embryonic stem cells and may have the potential to generate other types of cells as well. To achieve such reprogramming of adult stem cells it may be needed to fuse with cells from the target tissue, or to introduce specific genes. (see the section 'epigenetics' on the EUSEM server for a discussion of the principles of differentiation and programming)

Scientists in many laboratories are therefore trying to find better ways to grow large quantities of adult stem cells in cell culture and to manipulate them to generate induced pluripotent stem cells (iPSC), so they can be used to treat injury or disease. This would circumvent the ethical problems of using embryos for research and medical treatments. One such treatment has been used for 40 years already: adult hematopoietic, or blood-forming, stem cells from bone marrow have been used in hematopoietic stem cell transplantation (HSCT) for hematological malignancies (leukaemia patients). Examples of future treatments include regenerating bone using cells derived from bone marrow stroma, developing insulin-producing cells for type 1 diabetes and repairing damaged heart muscle following a heart attack with cardiac muscle cells.

The induced pluripotent stem (iPS) cell technology could even brought a step further if normal, fully differentiated adult cells could routinely be reprogrammed to act like pluripotent embryonic cells. Several studies have now reported successes using regular skin cells or cells from connective tissue. In these experiments the cells underwent genetic modification by the introduction of specific genes that are active in tumours. While the iPS cells display the necessary properties of embryonic stem cells, their genetic makeup is not considered as safe enough to use in treating diseases in patients.

Use your own stem cells   -^-

Future stem cell therapies probably would in majority rely on cells that are donated by another person. This raises the possibility of donor cell rejection by the patient's immune system. Some stem cells are more genetically compatible with the patient's own tissue than others and there are indications that some embryonic stem cells can be transplanted without any major rejection problems. It may also be possible to use a person's own source of stem cells to regenerate tissue, which would eliminate the danger of rejection. There are a number of options:

- Collection of healthy adult stem cells from a patient and manipulate the sample as needed for the transplant procedure (this is done with bone marrow).
- Generation of induced pluripotent stem cells using the patient's own tissue as starting material. This can be achieved by either reprogramming or by somatic cell nuclear transfer as used in reproductive cloning - Collection and storage until the moment when needed of stem cells originating from the patient's own umbilical cord blood.
- A so far hypothetical possibility is to manipulate and activate existing stem cells within the body, by using new drugs, to restore a lost function inside the patient's body.

Any stem cell therapy should be proven safe for the patient. Researchers have recognised that many common cancers may be caused by adult stem cells with damaged DNA, leading to a change in behaviour like uncontrolled growth, cell type change and migration. Just sampling stem cells from an individual and reintroduction in a tissue with defects may therefore turn out to be counterproductive (if helpful in the first place). Embryonic germ stem cells seem to be the safest source of stem cells in this respect, but these could in turn pose problems with immune rejection since these originate, by definition, from another individual.

Human gene therapy   -^-

There are many human diseases that are caused by faulty or missing genes. Scientists expect that, if the exact problem in the DNA sequence is known, it will (and in some cases it already has) become possible to either develop a cure for the patient, or detect the condition in early embryo's, or even alter egg or sperm cells in such a way that the genetic defect is corrected before the embryo is created. The latter possibility of course faces major ethical dilemma's.

Some diseases are relatively rare since they only occur if both mother's and father's genes have defects (autosomal recessive, i.e. cystic fibrosis), for others a single defective gene is sufficient to have the disease (autosomal dominant, i.e. Huntington's disease) and sometimes aberrations in number (trisomy, e.g. Down's syndrome) or missing (sex) chromosomes (monosomy, i.e. Turner syndrome) causes the disease.

Somatic cell gene therapy aims to introduce corrected DNA sequences in body cells. This has been shown possible when using disabled viruses that normally infect human cells. The technique has shown experimental successes in treating patients with cystic fibrosis who were given a genetically modified virus for inhalation. Since this virus was equipped with a functional human gene, that was missing in the patients, cells in the lungs acquired the possibility to synthesise a crucial protein that was missing before. This type of gene therapy only targets the non-reproductive body cells. Any changes introduced by the treatment will therefore not be transmitted to the next generation.

Inheritable genetic modification (IGM) is the type of gene therapy that would bring about changes in genes that would subsequently be passed on to future generations. It is also called germ-line therapy, since the genetic changes would be made in eggs, sperm or early embryos. It would be by far the most consequential type of genetic modification as it would open the door to irreversibly altering the human species. For this reason IGM has not been tried in humans and it is unlikely to be developed in the near future.

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