Genetically modified organism (updated)

From Wikipedia, the free encyclopedia

A genetically modified organism (GMO) is any organism whose genetic material has been altered using genetic engineering techniques (i.e., a genetically engineered organism). GMOs are used to produce many medications and genetically modified foods and are widely used in scientific research and the production of other goods. The term GMO is very close to the technical legal term, 'living modified organism', defined in the Cartagena Protocol on Biosafety, which regulates international trade in living GMOs (specifically, "any living organism that possesses a novel combination of genetic material obtained through the use of modern biotechnology").

A more specifically defined type of GMO is a "transgenic organism." This is an organism whose genetic makeup has been altered by the addition of genetic material from an unrelated organism. This should not be confused with the more general way in which "GMO" is used to classify genetically altered organisms, as typically GMOs are organisms whose genetic makeup has been altered without the addition of genetic material from an unrelated organism.

The first genetically modified mouse was created in 1974 by Rudolf Jaenisch, and the first plant was produced in 1983.

Production

A gene gun uses biolistics to insert DNA into plant tissue.

Creating a genetically modified organism (GMO) is a multi-step process. Genetic engineers must isolated the gene they wish to insert into the host organism. This can be taken from a cell containing the gene or artificially synthesised. If the chosen gene or the donor organism's genome has been well studied it may already be accessible from a genetic library. The gene is then combined with other genetic elements, including a promoter and terminator region and a selectable marker.

There are a number of techniques available for inserting the isolated gene into the host genome. Bacteria can be induced to take up foreign DNA by being exposed to certain stresses (e.g. thermal or electric shock). DNA is generally inserted into animal cells using microinjection, where it can be injected through the cell's nuclear envelope directly into the nucleus, or through the use of viral vectors. In plants the DNA is often inserted using Agrobacterium-mediated recombination, biolistics or electroporation.

As only a single cell is transformed with genetic material, the organism must be regenerated from that single cell. In plants this is accomplished through tissue culture. In animals it is necessary to ensure that the inserted DNA is present in the embryonic stem cells. Further testing using PCR, Southern hybridization, and DNA sequencing is conducted to confirm that an organism contains the new gene.

Traditionally the new genetic material was inserted randomly within the host genome. Gene targeting techniques, which creates double-stranded breaks and takes advantage on the cells natural homologous recombination repair systems, have been developed to target insertion to exact locations. Genome editing uses artificially engineered nucleases that create breaks at specific points. There are four families of engineered nucleases: meganucleases, zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and the Cas9-guideRNA system (adapted from CRISPR). TALEN and CRISPR are the two most commonly used and each has its own advantages. TALENs have greater target specificity, while CRISPR is easier to design and more efficient.

History

Herbert Boyer (pictured) and Stanley Cohen created the first genetically modified organism in 1973.

Humans have domesticated plants and animals since around 12,000 BCE, using selective breeding or artificial selection (as contrasted with natural selection). The process of selective breeding, in which organisms with desired traits (and thus with the desired genes) are used to breed the next generation and organisms lacking the trait are not bred, is a precursor to the modern concept of genetic modification. Various advancements in genetics allowed humans to directly alter the DNA and therefore genes of organisms. In 1972 Paul Berg created the first recombinant DNA molecule when he combined DNA from a monkey virus with that of the lambda virus.

Herbert Boyer and Stanley Cohen made the first genetically modified organism in 1973. They took a gene from a bacterium that provided resistance to the antibiotic kanamycin, inserted it into a plasmid and then induced another bacteria to incorporate the plasmid. The bacteria was then able to survive in the presence of kanamycin. Boyer and Cohen expressed other genes in bacteria. This included genes from the toad Xenopus laevis in 1974, creating the first GMO expressing a gene from an organism from different kingdom.

In 1974 Rudolf Jaenisch created the first GM animal.

In 1974 Rudolf Jaenisch created a transgenic mouse by introducing foreign DNA into its embryo, making it the world’s first transgenic animal. However it took another eight years before transgenic mice were developed that passed the transgene to their offspring. Genetically modified mice were created in 1984 that carried cloned oncogenes, predisposing them to developing cancer. Mice with genes knocked out (knockout mouse) were created in 1989. The first transgenic livestock were produced in 1985 and the first animal to synthesise transgenic proteins in their milk were mice, engineered to produce human tissue plasminogen activator in 1987.

In 1983 the first genetically engineered plant was developed by Michael W. Bevan, Richard B. Flavell and Mary-Dell Chilton. They infected tobacco with Agrobacterium transformed with an antibiotic resistance gene and through tissue culture techniques were able to grow a new plant containing the resistance gene. The gene gun was invented in 1987, allowing transformation of plants not susceptible to Agrobacterium infection. In 2000, Vitamin A-enriched golden rice, was the first plant developed with increased nutrient value.

In 1976 Genentech, the first genetic engineering company was founded by Herbert Boyer and Robert Swanson; a year later, the company produced a human protein (somatostatin) in E.coli. Genentech announced the production of genetically engineered human insulin in 1978. The insulin produced by bacteria, branded humulin, was approved for release by the Food and Drug Administration in 1982. In 1988 the first human antibodies were produced in plants. In 1987, the ice-minus strain of Pseudomonas syringae became the first genetically modified organism to be released into the environment when a strawberry field and a potato field in California were sprayed with it.

The first genetically modified crop, an antibiotic-resistant tobacco plant, was produced in 1982. China was the first country to commercialize transgenic plants, introducing a virus-resistant tobacco in 1992. In 1994 Calgene attained approval to commercially release the Flavr Savr tomato, the first genetically modified food. Also in 1994, the European Union approved tobacco engineered to be resistant to the herbicide bromoxynil, making it the first genetically engineered crop commercialized in Europe. An insect resistant Potato was approved for release in the US in 1995, and by 1996 approval had been granted to commercially grow 8 transgenic crops and one flower crop (carnation) in 6 countries plus the EU.

In 2010, scientists at the J. Craig Venter Institute, announced that they had created the first synthetic bacterial genome. They named it Synthia and it was the world's first synthetic life form.

The first genetically modified animal to be commercialised was the GloFish, a Zebra fish with a fluorescent gene added that allows it to glow in the dark under ultraviolet light. The first genetically modified animal to be approved for food use was AquAdvantage salmon in 2015. The salmon were transformed with a growth hormone-regulating gene from a Pacific Chinook salmon and a promoter from an ocean pout enabling it to grow year-round instead of only during spring and summer.

Types

There are a wide variety of organisms that have been genetically engineered, from animals to plants and microorganisms. Genes have been transferred within the same species, across species and even across kingdoms. New genes can be introduced, or endogenous genes can be enhanced, altered or knocked out. GMOs have been used in biological and medical research, production of pharmaceutical drugs, experimental medicine (e.g. gene therapy and vaccines against the Ebola virus), and agriculture (e.g. golden rice, resistance to herbicides), with developing uses in conservation.

Microorganisms

Bacteria

Bacteria were the first organisms to be genetically modified in the laboratory, due to the relative ease of modifying their chromosomes. This ease made them important tools for the creation of other GMOs. Genes and other genetic information from a wide range of organisms can be added to a plasmid and inserted into bacteria for storage and modification. Bacteria are cheap, easy to grow, clonal, multiply quickly, are relatively easy to transform, and can be stored at −80 °C almost indefinitely. Once a gene is isolated it can be stored inside the bacteria, providing an unlimited supply for research. The large number of custom plasmids make manipulating DNA excised from bacteria relatively easy. In the field of synthetic biology, they have been used to test various synthetic approaches, from synthesizing genomes to creating novel nucleotides.

Bacteria have been used in the production of food for a long time, and specific strains have been developed and selected for that work on an industrial scale. They can be used to produce enzymes, amino acids, flavourings, and other compounds used in food production. With the advent of genetic engineering, new genetic changes can easily be introduced into these bacteria. Most food-producing bacteria are lactic acid bacteria, and this is where the majority of research into genetically engineering food-producing bacteria has gone. The bacteria can be modified to operate more efficiently, reduce toxic byproduct production, increase output, create improved compounds, and remove unnecessary pathways. Food products from genetically modified bacteria include alpha-amylase, which converts starch to simple sugars, chymosin, which clots milk protein for cheese making, and pectinesterase, which improves fruit juice clarity.

Genetically modified bacteria are used to produce large amounts of proteins for industrial use. Generally the bacteria are grown to a large volume before the gene encoding the protein is activated. The bacteria are then harvested and the desired protein purified from them. The high cost of extraction and purification has meant that only high value products have been produced at an industrial scale. The majority of these products are human proteins for use in medicine. Many of these proteins are impossible or difficult to obtain via natural methods and they are less likely to be contaminated with pathogens, making them safer. The first medicinal use of GM bacteria was to produce the protein insulin to treat diabetes. Other medicines produced include clotting factors to treat haemophilia, human growth hormone to treat various forms of dwarfism, interferon to treat some cancers, erythropoietin for anemic patients, and tissue plasminogen activator which dissolves blood clots. Outside of medicine they have been used to produce biofuels. There is interest in developing an extracellular expression system within the bacteria to reduce costs and make the production of more products economical.

With greater understanding of the role that the micobiome plays in human health, there is the potential to treat diseases by genetically altering the bacteria to, themselves, be therapeutic agents. Ideas include altering gut bacteria so they destroy harmful bacteria, or using bacteria to replace or increase deficient enzymes or proteins. One research focus is to modify Lactobacillus, bacteria that naturally provide some protection against HIV, with genes that will further enhance this protection. If the bacteria do not form colonies inside the patient, the person must repeatedly ingest the modified bacteria in order to get the required doses. Enabling the bacteria to form a colony could provide a more long-term solution, but could also raise safety concerns as interactions between bacteria and the human body are less well understood than with traditional drugs. There are concerns that horizontal gene transfer to other bacteria could have unknown effects. As of 2018 there are clinical trials underway testing the efficacy and safety of these treatments.

For over a century bacteria have been used in agriculture. Crops have been inoculated with Rhizobia (and more recently Azospirillum) to increase their production or to allow them to be grown outside their original habitat. Application of Bacillus thuringiensis (Bt) and other bacteria can help protect crops from insect infestation and plant diseases. With advances in genetic engineering, these bacteria have been manipulated for increased efficiency and expanded host range. Markers have also been added to aid in tracing the spread of the bacteria. The bacteria that naturally colonise certain crops have also been modified, in some cases to express the Bt genes responsible for pest resistance. Pseudomonas strains of bacteria cause frost damage by nucleating water into ice crystals around themselves. This led to the development of ice-minus bacteria, that have the ice-forming genes removed. When applied to crops they can compete with the ice-plus bacteria and confer some frost resistance.

This artwork is made with bacteria modified to express 8 different colours of fluorescent proteins.

Other uses for genetically modified bacteria include bioremediation, where the bacteria are used to convert pollutants into a less toxic form. Genetic engineering can increase the levels of the enzymes used to degrade a toxin or to make the bacteria more stable under environmental conditions. Bioart has also been created using genetically modified bacteria. In the 1980s artist Jon Davis and geneticist Dana Boyd converted the Germanic symbol for femininity (ᛉ) into binary code and then into a DNA sequence, which was then expressed in Escherichia coli. This was taken a step further in 2012, when a whole book was encoded onto DNA. Paintings have also been produced using bacteria transformed with fluorescent proteins.

Virus

Viruses are often modified so they can be used as vectors for inserting genetic information into other organisms. This process is called transduction and if successful the recipient of the introduced DNA becomes a GMO.
In 2017 researchers genetically modified a virus to express spinach defensin proteins. The virus was injected into orange trees to combat citrus greening disease that had reduced orange production 70% since 2005.

Yeast

As of 2016 two genetically modified yeasts involved in the fermentation of wine have been commercialised. One has increased malolactic fermentation efficiency, while the other prevents the production of dangerous ethyl carbamate compounds during fermentation.

Plants

Kenyans examining insect-resistant transgenic Bt corn

Transgenic plants have been engineered for scientific research, to create new colours in plants, and to create different crops.

In research, plants are engineered to help discover the functions of certain genes. One way to do this is to knock out the gene of interest and see what phenotype develops. Another strategy is to attach the gene to a strong promoter and see what happens when it is over expressed. A common technique used to find out where the gene is expressed is to attach it to GUS or a similar reporter gene that allows visualisation of the location.
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Suntory "blue" rose

After thirteen years of collaborative research, an Australian company – Florigene, and a Japanese company – Suntory, created a blue rose (actually lavender or mauve) in 2004. The genetic engineering involved three alterations – adding two genes, and interfering with another. One of the added genes was for the blue plant pigment delphinidin cloned from the pansy. The researchers then used RNA interference (RNAi) technology to depress all color production by endogenous genes by blocking a crucial protein in color production, called dihydroflavonol 4-reductase (DFR), and adding a variant of that protein that would not be blocked by the RNAi but that would allow the delphinidin to work. The roses are sold in Japan, the United States, and Canada. Florigene has also created and sells lavender-colored carnations that are genetically engineered in a similar way.

Simple plants and plant cells have been genetically engineered for production of biopharmaceuticals in bioreactors as opposed to cultivating plants in open fields. Work has been done with duckweed Lemna minor, the algae Chlamydomonas reinhardtii and the moss Physcomitrella patens. An Israeli company, Protalix, has developed a method to produce therapeutics in cultured transgenic carrot and tobacco cells. Protalix and its partner, Pfizer, received FDA approval to market its drug Elelyso, a treatment for Gaucher's disease, in 2012.

Crops

Genetically modified crops (GM crops, or biotech crops) are plants used in agriculture, the DNA of which has been modified using genetic engineering techniques. In most cases the aim is to introduce a new trait to the plant which does not occur naturally in the species. Examples in food crops include resistance to certain pests, diseases, or environmental conditions, reduction of spoilage, or resistance to chemical treatments (e.g. resistance to a herbicide), or improving the nutrient profile of the crop. Examples in non-food crops include production of pharmaceutical agents, biofuels, and other industrially useful goods, as well as for bioremediation.

Farmers have widely adopted GM technology. Between 1996 and 2013, the total surface area of land cultivated with GM crops increased by a factor of 100, from 17,000 square kilometers (4,200,000 acres) to 1,750,000 km² (432 million acres). 10% of the world's croplands were planted with GM crops in 2010. In the US, by 2014, 94% of the planted area of soybeans, 96% of cotton and 93% of corn were genetically modified varieties. In recent years, GM crops expanded rapidly in developing countries. In 2013, approximately 18 million farmers grew 54% of worldwide GM crops in developing countries.

Cisgenic plants

Cisgenesis, sometimes also called intragenesis, is a product designation for a category of genetically engineered plants. A variety of classification schemes have been proposed that order genetically modified organisms based on the nature of introduced genotypical changes rather than the process of genetic engineering.

While some genetically modified plants are developed by the introduction of a gene originating from distant, sexually incompatible species into the host genome, cisgenic plants contain genes that have been isolated either directly from the host species or from sexually compatible species. The new genes are introduced using recombinant DNA methods and gene transfer. Some scientists hope that the approval process of cisgenic plants might be simpler than that of proper transgenics, but it remains to be seen.

Conservation

Genetically modified organisms have been proposed to aid conservation of plant species threatened by extinction. Many trees face the threat of invasive plants and diseases, such as the emerald ash borer in North American and the fungal disease, Ceratocystis platani, in European plane trees. A suggested solution to increase the resilience of threatened tree species is to genetically modify individuals by transferring resistant genes. Papaya trees are an example of a species that was successfully conserved using genetic modification. The papaya ringspot virus (PRSV) devastated papaya trees in Hawaii in the twentieth century until transgenic papaya plants were given pathogen-derived resistance.

However, genetic modification for conservation in plants remains mainly speculative and further experimentation is needed before the technique can be widely implemented. A main concern with using genetic modification for conservation purposes is that a transgenic species may no longer bear enough resemblance to the original species to truly claim that the original species is being conserved. Instead, the transgenic species may be genetically different enough to be considered a new species, thus diminishing the conservation worth of genetic modification.

Mammals

Some chimeras, like the blotched mouse shown, are created through genetic modification techniques like gene targeting.

Genetically modified mammals are an important category of genetically modified organisms. Ralph L. Brinster and Richard Palmiter developed the techniques responsible for transgenic mice, rats, rabbits, sheep, and pigs in the early 1980s, and established many of the first transgenic models of human disease, including the first carcinoma caused by a transgene. The process of genetically engineering animals is a slow, tedious, and expensive process. However, new technologies are making genetic modifications easier and more precise.

The first transgenic (genetically modified) animal was produced by injecting DNA into mouse embryos then implanting the embryos in female mice.

Genetically modified animals currently being developed can be placed into six different broad classes based on the intended purpose of the genetic modification:

1. to research human diseases (for example, to develop animal models for these diseases);
2. to produce industrial or consumer products (fibres for multiple uses);
3. to produce products intended for human therapeutic use (pharmaceutical products or tissue for implantation);
4. to enrich or enhance the animals' interactions with humans (hypo-allergenic pets);
5. to enhance production or food quality traits (faster growing fish, pigs that digest food more efficiently);
6. to improve animal health (disease resistance).

Research use

Dolly was a female domestic sheep and the first animal to be cloned from an adult somatic cell.

Transgenic animals are used as experimental models to perform phenotypic and for testing in biomedical research.

Genetically modified (genetically engineered) animals are becoming more vital to the discovery and development of cures and treatments for many serious diseases. By altering the DNA or transferring DNA to an animal, we can develop certain proteins that may be used in medical treatment. Stable expressions of human proteins have been developed in many animals, including sheep, pigs, and rats. Human-alpha-1-antitrypsin, which has been tested in sheep and is used in treating humans with this deficiency and transgenic pigs with human-histo-compatibility have been studied in the hopes that the organs will be suitable for transplant with less chances of rejection.

Scientists have genetically engineered several organisms, including some mammals, to include green fluorescent protein (GFP), first observed in the jellyfish, Aequorea victoria in 1962, for medical research purposes (Chalfie, Shimoura, and Tsien were awarded the Nobel prize in Chemistry in 2008 for the discovery and development of GFP). For example, fluorescent pigs have been bred to study human organ transplants (xenotransplantation), regenerating ocular photoreceptor cells, and other topics. In 2011 a Japanese-American team created green-fluorescent cats to find therapies for HIV/AIDS and other diseases as feline immunodeficiency virus (FIV) is related to HIV.

In 2009, scientists in Japan announced that they had successfully transferred a gene into a primate species (marmosets) and produced a stable line of breeding transgenic primates for the first time. Their first research target for these marmosets was Parkinson's disease, but they were also considering amyotrophic lateral sclerosis and Huntington's disease.

Human therapeutics and xenotransplants

Herman the Bull, Naturalis, for the production of lactoferrin enhanced milk

 

Transgenic pig for cheese production

Within the field known as pharming, intensive research has been conducted to develop transgenic animals that produce biotherapeutics. On 6 February 2009, the U.S. Food and Drug Administration approved the first human biological drug produced from such an animal, a goat. The drug, ATryn, is an anticoagulant which reduces the probability of blood clots during surgery or childbirth. It is extracted from the goat's milk.

Some animals are also genetically modified so that they can provide organs that are suitable and safe to transplant into humans (xenotransplants). An example are pigs that are genetically modified so that their organs can no longer carry retroviruses (which can pose a danger to humans, when transplanted into them). Other genetically modified pigs have had alpha galactosidase transferase knocked out and fortified with hCD46 and the hTM molecule. Pig lungs from genetically modified pigs for instance are already being considered for transplantation into humans. Besides use of genetic modification to allow the providing of safer animal organs for transplantation, genetic modification can also be used to allow the animal to grow human organs inside their body. Such animals, which are hence composed of a mixture of cells from more than one species, are called "chimeras." One project, undertaken by Pablo Ross of the University of California, involves the growing of a human pancreas inside a pig.

Food quality traits

In 2006, a pig was engineered to produce omega-3 fatty acids through the expression of a roundworm gene.

Enviropig was a genetically enhanced line of Yorkshire pigs in Canada created with the capability of digesting plant phosphorus more efficiently than conventional Yorkshire pigs. The project ended in 2012. These pigs produced the enzyme phytase, which breaks down the indigestible phosphorus, in their saliva. The enzyme was introduced into the pig chromosome by pronuclear microinjection. With this enzyme, the animal is able to digest cereal grain phosphorus. The use of these pigs would reduce the potential of water pollution since they excrete from 30 to 70.7% less phosphorus in manure depending upon the age and diet. The lower concentrations of phosphorus in surface runoff reduces algal growth, because phosphorus is the limiting nutrient for algae. Because algae consume large amounts of oxygen, it can result in dead zones for fish.

In 2011, Chinese scientists generated dairy cows genetically engineered with genes from human beings to produce milk that would be the same as human breast milk. This could potentially benefit mothers who cannot produce breast milk but want their children to have breast milk rather than formula. Aside from milk production, the researchers claim these transgenic cows to be identical to regular cows. Two months later scientists from Argentina presented Rosita, a transgenic cow incorporating two human genes, to produce milk with similar properties as human breast milk. In 2012, researchers from New Zealand also developed a genetically engineered cow that produced allergy-free milk.

Goats have been genetically engineered to produce milk with strong spiderweb-like silk proteins in their milk.

Human gene therapy

Gene therapy, uses genetically modified viruses to deliver genes which can cure disease in humans. Although gene therapy is still relatively new, it has had some successes. It has been used to treat genetic disorders such as severe combined immunodeficiency, and Leber's congenital amaurosis. Treatments are also being developed for a range of other currently incurable diseases, such as cystic fibrosis, sickle cell anemia, Parkinson's disease, cancer, diabetes, heart disease and muscular dystrophy.

Conservation use

Genetically modified organisms have been used to conserve European wild rabbits in the Iberian peninsula and Australia. In both cases, the genetically modified organism used was a myxoma virus, but for opposite purposes: to protect the endangered population in Europe with immunizations and to regulate the overabundant population in Australia with contraceptives.

In the Iberian peninsula, the European wild rabbit population has experienced a sharp decline from viral diseases and overhunting. To protect the species from viral diseases, the myxoma virus was genetically modified to immunize the rabbits. The European wild rabbit population in Australia faces the opposite problem: lack of natural predators has made the introduced species invasive. The same myxoma virus was genetically modified to lower fertility in the Australian rabbit population.

Fish

Genetically modified fish are used for scientific research and as pets, and are being considered for use as food and as aquatic pollution sensors.
GM fish are widely used in basic research in genetics and development. Two species of fish, zebrafish and medaka, are most commonly modified because they have optically clear chorions (membranes in the egg), rapidly develop, and the 1-cell embryo is easy to see and microinject with transgenic DNA.

The GloFish is a patented brand of genetically modified (GM) fluorescent zebrafish with bright red, green, and orange fluorescent color. Although not originally developed for the ornamental fish trade, it became the first genetically modified animal to become publicly available as a pet when it was introduced for sale in 2003. They were quickly banned for sale in California.

GM fish have been developed with promoters driving an over-production of "all fish" growth hormone for use in the aquaculture industry to increase the speed of development and potentially reduce fishing pressure on wild stocks. This has resulted in dramatic growth enhancement in several species, including salmon, trout, and tilapia. AquaBounty Technologies, a biotechnology company working on bringing a GM salmon to market, claims that their GM AquAdvantage salmon can mature in half the time as wild salmon. AquaBounty applied for regulatory approval to market their GM salmon in the US, and was approved in November 2015. On 25 November 2013 Canada approved commercial scale production and export of GM Salmon eggs but they are not approved for human consumption in Canada.

Several academic groups have been developing GM zebrafish to detect aquatic pollution. The lab that originated the GloFish discussed above originally developed them to change color in the presence of pollutants, to be used as environmental sensors. A lab at University of Cincinnati has been developing GM zebrafish for the same purpose, as has a lab at Tulane University.

Recent research on pain in fish has resulted in concerns being raised that genetic-modifications induced for scientific research may have detrimental effects on the welfare of fish.

Frogs

Genetically modified frogs are used for scientific research and are widely used in basic research including genetics and early development. Two species of frog, Xenopus laevis and Xenopus tropicalis, are most commonly used.

GM frogs are also being used as pollution sensors, especially for endocrine disrupting chemicals.

Invertebrates

Fruit flies

In biological research, transgenic fruit flies (Drosophila melanogaster) are model organisms used to study the effects of genetic changes on development. Fruit flies are often preferred over other animals due to their short life cycle, low maintenance requirements, and relatively simple genome compared to many vertebrates.

Mosquitoes

In 2010, scientists created "malaria-resistant mosquitoes" in the laboratory. The World Health Organization estimated that malaria killed almost one million people in 2008. Genetically modified male mosquitoes containing a lethal gene have been developed to combat the spread of dengue fever and the Zika virus. Aedes aegypti mosquitoes, the single most important carrier of dengue fever and the Zika virus, were reduced by 80% in a 2010 trial of these GM mosquitoes in the Cayman Islands and by 90% in a 2015 trial in Bahia, Brazil. In comparison, the Florida Keys Mosquito Control District has achieved only 30–60% population reduction with traps and pesticide spraying. In 2016 FDA approved a genetically modified mosquito intervention for Key West, Florida. UK firm Oxitec proposed the release of millions of modified male (non-biting) mosquitoes to compete with wild males for mates. The males are engineered so that their offspring die before maturing, helping to eradicate mosquito-borne disease. Final approval was to be based on a local referendum to be held in November. Andrea Crisanti, a molecular biologist at Imperial College in London is working on ways to stop the A. gambiae mosquito from transmitting disease.

Bollworms

A strain of Pectinophora gossypiella (Pink bollworm) has been genetically engineered to express a red fluorescent protein. This allows researchers to monitor bollworms that have been sterilized by radiation and released to reduce bollworm infestation. The strain has been field tested for over three years and has been approved for release.

Cnidaria

Cnidaria such as Hydra and the sea anemone Nematostella vectensis are attractive model organisms to study the evolution of immunity and certain developmental processes. An important technical breakthrough was the development of procedures for generation of stable transgenic hydras and sea anemones by embryo microinjection.

Regulation

The regulation of genetic engineering concerns the approaches taken by governments to assess and manage the risks associated with the use of genetic engineering technology and the development and release of genetically modified organisms (GMO), including genetically modified crops and genetically modified fish. There are differences in the regulation of GMOs between countries, with some of the most marked differences occurring between the USA and Europe. Regulation varies in a given country depending on the intended use of the products of the genetic engineering. For example, a crop not intended for food use is generally not reviewed by authorities responsible for food safety. The European Union differentiates between approval for cultivation within the EU and approval for import and processing. While only a few GMOs have been approved for cultivation in the EU a number of GMOs have been approved for import and processing. The cultivation of GMOs has triggered a debate about the market for GMOs in Europe. Depending on the coexistence regulations, incentives for cultivation of GM crops differ.

Controversy

There is controversy over GMOs, especially with regard to their use in producing food. The dispute involves buyers, biotechnology companies, governmental regulators, nongovernmental organizations, and scientists. The key areas of controversy related to GMO food are whether GM food should be labeled, the role of government regulators, the effect of GM crops on health and the environment, the effect on pesticide resistance, the impact of GM crops for farmers, and the role of GM crops in feeding the world population. In 2014, sales of products that had been labeled as non-GMO grew 30 percent to $1.1 billion.

There is a scientific consensus that currently available food derived from GM crops poses no greater risk to human health than conventional food, but that each GM food needs to be tested on a case-by-case basis before introduction. Nonetheless, members of the public are much less likely than scientists to perceive GM foods as safe. The legal and regulatory status of GM foods varies by country, with some nations banning or restricting them, and others permitting them with widely differing degrees of regulation.

No reports of ill effects have been proven in the human population from ingesting GM food. Although labeling of GMO products in the marketplace is required in many countries, it is not required in the United States and no distinction between marketed GMO and non-GMO foods is recognized by the US FDA. In a May 2014 article in The Economist it was argued that, while GM foods could potentially help feed 842 million malnourished people globally, laws such as the one passed in Vermont, to require labeling of foods containing genetically modified ingredients, could have the unintended consequence of interrupting the process of spreading GM technologies to impoverished countries that suffer with food security problems.

The Organic Consumers Association, and the Union of Concerned Scientists, and Greenpeace stated that risks have not been adequately identified and managed, and they have questioned the objectivity of regulatory authorities. Some health groups say there are unanswered questions regarding the potential long-term impact on human health from food derived from GMOs, and propose mandatory labeling or a moratorium on such products. Concerns include contamination of the non-genetically modified food supply, effects of GMOs on the environment and nature, the rigor of the regulatory process, and consolidation of control of the food supply in companies that make and sell GMOs, or concerns over the use of herbicides with glyphosate.

In order to address some of these concerns GMOs have been developed with traits to help control their spread. This includes bacteria modified to depend on nutrients that cannot be found in nature and developing genetic use restriction technology that causes the second generation of GM plants to be sterile.

Biological patenting

The privatization of GM patenting is controversial because once genetic sequences are patented, farmers of GM foods are often forced to pay fees for their harvest. One example is from 1998, when RiceTec patented a GM version of basmati rice. Due to the World Trade Organization's bans on "barriers" to trade, it was prohibited for GMOs to be labeled as such. Though RiceTec illegally accessed the Filipino genetic data bank that made their discoveries possible and therefore patentable, and the genes were copied from basmati rice already being grown in the Philippines, these GM seeds were sold throughout the region, and Filipino farmers were fined for harvesting a plant they had been growing for free previously.

Artificial gene synthesis

From Wikipedia, the free encyclopedia

DNA Double Helix

Artificial gene synthesis, sometimes known as DNA printing is a method in synthetic biology that is used to create artificial genes in the laboratory. Based on solid-phase DNA synthesis, it differs from molecular cloning and polymerase chain reaction (PCR) in that it does not have to begin with preexisting DNA sequences. Therefore, it is possible to make a completely synthetic double-stranded DNA molecule with no apparent limits on either nucleotide sequence or size.

The method has been used to generate functional bacterial or yeast chromosomes containing approximately one million base pairs. Creating novel nucleobase pairs in addition to the two base pairs in nature could greatly expand the genetic code.

Synthesis of the first complete gene, a yeast tRNA, was demonstrated by Har Gobind Khorana and coworkers in 1972. Synthesis of the first peptide- and protein-coding genes was performed in the laboratories of Herbert Boyer and Alexander Markham, respectively.

Commercial gene synthesis services are now available. Approaches are most often based on a combination of organic chemistry and molecular biology techniques and entire genes may be synthesized "de novo", without the need for template DNA. Gene synthesis is an important tool in many fields of recombinant DNA technology including heterologous gene expression, vaccine development, gene therapy and molecular engineering. The synthesis of nucleic acid sequences can be more economical than classical cloning and mutagenesis procedures. It is also a powerful and flexible engineering tool for creating and designing new DNA sequences and protein functions.

Gene optimization

While the ability to make increasingly long stretches of DNA efficiently and at lower prices is a technological driver of this field, increasingly attention is being focused on improving the design of genes for specific purposes. Early in the genome sequencing era, gene synthesis was used as an (expensive) source of cDNAs that were predicted by genomic or partial cDNA information but were difficult to clone. As higher quality sources of sequence verified cloned cDNA have become available, this practice has become less urgent.

Producing large amounts of protein from gene sequences (or at least the protein coding regions of genes, the open reading frame) found in nature can sometimes prove difficult and is a problem of sufficient impact that scientific conferences have been devoted to the topic. Many of the most interesting proteins sought by molecular biologists are normally regulated to be expressed in very low amounts in wild type cells. Redesigning these genes offers a means to improve gene expression in many cases. Rewriting the open reading frame is possible because of the degeneracy of the genetic code. Thus it is possible to change up to about a third of the nucleotides in an open reading frame and still produce the same protein. The available number of alternate designs possible for a given protein is astronomical. For a typical protein sequence of 300 amino acids there are over 10¹⁵⁰ codon combinations that will encode an identical protein. Using optimization methods such as replacing rarely used codons with more common codons sometimes have dramatic effects. Further optimizations such as removing RNA secondary structures can also be included. At least in the case of E. coli, protein expression is maximized by predominantly using codons corresponding to tRNA that retain amino acid charging during starvation. Computer programs written to perform these, and other simultaneous optimizations are used to handle the enormous complexity of the task. A well optimized gene can improve protein expression 2 to 10 fold, and in some cases more than 100 fold improvements have been reported. Because of the large numbers of nucleotide changes made to the original DNA sequence, the only practical way to create the newly designed genes is to use gene synthesis.

Standard methods

Oligonucleotide synthesis

Oligonucleotides are chemically synthesized using building blocks called nucleoside phosphoramidites. These can be normal or modified nucleosides which have protecting groups to prevent their amines, hydroxyl groups and phosphate groups from interacting incorrectly. One phosphoramidite is added at a time, the 5' hydroxyl group is deprotected and a new base is added and so on. The chain grows in the 3' to 5' direction, which is backwards relative to biosynthesis. At the end, all the protecting groups are removed. Nevertheless, being a chemical process, several incorrect interactions occur leading to some defective products. The longer the oligonucleotide sequence that is being synthesized, the more defects there are, thus this process is only practical for producing short sequences of nucleotides. The current practical limit is about 200 bp (base pairs) for an oligonucleotide with sufficient quality to be used directly for a biological application. HPLC can be used to isolate products with the proper sequence. Meanwhile, a large number of oligos can be synthesized in parallel on gene chips. For optimal performance in subsequent gene synthesis procedures they should be prepared individually and in larger scales.

Annealing based connection of oligonucleotides

Usually, a set of individually designed oligonucleotides is made on automated solid-phase synthesizers, purified and then connected by specific annealing and standard ligation or polymerase reactions. To improve specificity of oligonucleotide annealing, the synthesis step relies on a set of thermostable DNA ligase and polymerase enzymes. To date, several methods for gene synthesis have been described, such as the ligation of phosphorylated overlapping oligonucleotides, the Fok I method and a modified form of ligase chain reaction for gene synthesis. Additionally, several PCR assembly approaches have been described. They usually employ oligonucleotides of 40-50 nucleotides long that overlap each other. These oligonucleotides are designed to cover most of the sequence of both strands, and the full-length molecule is generated progressively by overlap extension (OE) PCR, thermodynamically balanced inside-out (TBIO) PCR or combined approaches. The most commonly synthesized genes range in size from 600 to 1,200 bp although much longer genes have been made by connecting previously assembled fragments of under 1,000 bp. In this size range it is necessary to test several candidate clones confirming the sequence of the cloned synthetic gene by automated sequencing methods.

Limitations

Moreover, because the assembly of the full-length gene product relies on the efficient and specific alignment of long single stranded oligonucleotides, critical parameters for synthesis success include extended sequence regions comprising secondary structures caused by inverted repeats, extraordinary high or low GC-content, or repetitive structures. Usually these segments of a particular gene can only be synthesized by splitting the procedure into several consecutive steps and a final assembly of shorter sub-sequences, which in turn leads to a significant increase in time and labor needed for its production. The result of a gene synthesis experiment depends strongly on the quality of the oligonucleotides used. For these annealing based gene synthesis protocols, the quality of the product is directly and exponentially dependent on the correctness of the employed oligonucleotides. Alternatively, after performing gene synthesis with oligos of lower quality, more effort must be made in downstream quality assurance during clone analysis, which is usually done by time-consuming standard cloning and sequencing procedures. Another problem associated with all current gene synthesis methods is the high frequency of sequence errors because of the usage of chemically synthesized oligonucleotides. The error frequency increases with longer oligonucleotides, and as a consequence the percentage of correct product decreases dramatically as more oligonucleotides are used. The mutation problem could be solved by shorter oligonucleotides used to assemble the gene. However, all annealing based assembly methods require the primers to be mixed together in one tube. In this case, shorter overlaps do not always allow precise and specific annealing of complementary primers, resulting in the inhibition of full length product formation. Manual design of oligonucleotides is a laborious procedure and does not guarantee the successful synthesis of the desired gene. For optimal performance of almost all annealing based methods, the melting temperatures of the overlapping regions are supposed to be similar for all oligonucleotides. The necessary primer optimization should be performed using specialized oligonucleotide design programs. Several solutions for automated primer design for gene synthesis have been presented so far.

Error correction procedures

To overcome problems associated with oligonucleotide quality several elaborate strategies have been developed, employing either separately prepared fishing oligonucleotides, mismatch binding enzymes of the mutS family or specific endonucleases from bacteria or phages. Nevertheless, all these strategies increase time and costs for gene synthesis based on the annealing of chemically synthesized oligonucleotides.

Massively parallel sequencing has also been used as a tool to screen complex oligonucleotide libraries and enable the retrieval of accurate molecules. In one approach, oligonucleotides are sequenced on the 454 pyrosequencing platform and a robotic system images and picks individual beads corresponding to accurate sequence. In another approach, a complex oligonucleotide library is modified with unique flanking tags before massively parallel sequencing. Tag-directed primers then enable the retrieval of molecules with desired sequences by dial-out PCR.

Increasingly, genes are ordered in sets including functionally related genes or multiple sequence variants on a single gene. Virtually all of the therapeutic proteins in development, such as monoclonal antibodies, are optimized by testing many gene variants for improved function or expression.

Applications

Major applications of synthetic genes include synthesis of DNA sequences identified by high throughput sequencing but never cloned into plasmids and the ability to safely obtain genes for vaccine research without the need to grow the full pathogens. Digital manipulation of digital genetic code before synthesis into DNA can be used to optimize protein expression in a particular host, or remove non-functional segments in order to facilitate further replication of the DNA.

Synthesis of DNA allows DNA digital data storage.

DNA synthesis and synthetic biology

The significant drop in cost of gene synthesis in recent years due to increasing competition of companies providing this service has led to the ability to produce entire bacterial plasmids that have never existed in nature. The field of synthetic biology utilizes the technology to produce synthetic biological circuits, which are stretches of DNA manipulated to change gene expression within cells and cause the cell to produce a desired product.

Entire bacterial genomes

Synthia and Mycoplasma laboratorium

On June 28, 2007, a team at the J. Craig Venter Institute published an article in Science Express, saying that they had successfully transplanted the natural DNA from a Mycoplasma mycoides bacterium into a Mycoplasma capricolum cell, creating a bacterium which behaved like a M. mycoides.

On Oct 6, 2007, Craig Venter announced in an interview with UK's The Guardian newspaper that the same team had synthesized a modified version of the single chromosome of Mycoplasma genitalium using chemicals. The chromosome was modified to eliminate all genes which tests in live bacteria had shown to be unnecessary. The next planned step in this minimal genome project is to transplant the synthesized minimal genome into a bacterial cell with its old DNA removed; the resulting bacterium will be called Mycoplasma laboratorium. The next day the Canadian bioethics group, ETC Group issued a statement through their representative, Pat Mooney, saying Venter's "creation" was "a chassis on which you could build almost anything". The synthesized genome had not yet been transplanted into a working cell.

On May 21, 2010, Science reported that the Venter group had successfully synthesized the genome of the bacterium Mycoplasma mycoides from a computer record, and transplanted the synthesized genome into the existing cell of a Mycoplasma capricolum bacterium that had its DNA removed. The "synthetic" bacterium was viable, i.e. capable of replicating billions of times. The team had originally planned to use the M. genitalium bacterium they had previously been working with, but switched to M. mycoides because the latter bacterium grows much faster, which translated into quicker experiments. Venter describes it as "the first species.... to have its parents be a computer". The transformed bacterium is dubbed "Synthia" by ETC. A Venter spokesperson has declined to confirm any breakthrough at the time of this writing.

Yeast chromosome

In March 2014, Jef Boeke of the Langone Medical Centre at New York University, published that his team has synthesized one of the S. cerevisiae 16 yeast chromosomes, the chromosome III, that he named synIII. The procedure involved replacing the genes in the original chromosome with synthetic versions and the finished human made chromosome was then integrated into a yeast cell. It required designing and creating 273,871 base pairs of DNA – fewer than the 316,667 pairs in the original chromosome. In March 2017 6 yeast chromosomes were reported to have been synthesized.

Unnatural base pair (UBP)

DNA sequences have been described which use newly created nucleobases to form a third base pair, in addition to the two base pairs found in nature, A-T (adenine – thymine) and G-C (guanine – cytosine). Multiple research groups have been searching for a third base pair for DNA, including teams led by Steven A. Benner, Philippe Marliere, and Ichiro Hirao. Some new base pairs have been reported.

In 2012, a group of American scientists led by Floyd Romesberg, a chemical biologist at the Scripps Research Institute in San Diego, California, published that his team designed an unnatural base pair (UBP). The two new artificial nucleotides or Unnatural Base Pair (UBP) were named d5SICS and dNaM. More technically, these artificial nucleotides bearing hydrophobic nucleobases, feature two fused aromatic rings that form a (d5SICS–dNaM) complex or base pair in DNA. In 2014 the same team from the Scripps Research Institute reported that they synthesized a stretch of circular DNA known as a plasmid containing natural T-A and C-G base pairs along with the best-performing UBP Romesberg's laboratory had designed, and inserted it into cells of the common bacterium E. coli that successfully replicated the unnatural base pairs through multiple generations. This is the first known example of a living organism passing along an expanded genetic code to subsequent generations. This was in part achieved by the addition of a supportive algal gene that expresses a nucleotide triphosphate transporter which efficiently imports the triphosphates of both d5SICSTP and dNaMTP into E. coli bacteria. Then, the natural bacterial replication pathways use them to accurately replicate the plasmid containing d5SICS–dNaM.

The successful incorporation of a third base pair is a significant breakthrough toward the goal of greatly expanding the number of amino acids which can be encoded by DNA, from the existing 20 amino acids to a theoretically possible 172, thereby expanding the potential for living organisms to produce novel proteins. The artificial strings of DNA do not encode for anything yet, but scientists speculate they could be designed to manufacture new proteins which could have industrial or pharmaceutical uses.