Genetic engineering
Genetic engineering, also called genetic modification, is the direct manipulation of an organism's genome using biotechnology. (Indirect genetic modification through artificial selection has been practiced for centuries.) New DNA may be inserted in the host genome by first isolating and copying the genetic material of interest using molecular cloning methods to generate a DNA sequence, or by synthesizing the DNA, and then inserting this construct into the host organism. Genes may be removed, or "knocked out", using a nuclease. Gene targeting is a different technique that uses homologous recombination to change an endogenous gene, and can be used to delete a gene, remove exons, add a gene, or introduce point mutations.
An organism that is generated through genetic engineering is considered to be a genetically modified organism (GMO). The first GMOs were bacteria in 1973; GM mice were generated in 1974. Insulin-producing bacteria were commercialized in 1982 and genetically modified food has been sold since 1994. Glofish, the first GMO designed as a pet, was first sold in the United States December in 2003.
Genetic engineering techniques have been applied in numerous fields including research, agriculture, industrial biotechnology, and medicine. Enzymes used in laundry detergent and medicines such as insulin and human growth hormone are now manufactured in GM cells, experimental GM cell lines and GM animals such as mice or zebrafish are being used for research purposes, and genetically modified crops have been commercialized.
Definition
Genetic engineering alters the genetic makeup of an organism using techniques that remove heritable material or that introduce DNA prepared outside the organism either directly into the host or into a cell that is then fused or hybridized with the host.This involves using recombinant nucleic acid (DNA or RNA) techniques to form new combinations of heritable genetic material followed by the incorporation of that material either indirectly through a vector system or directly through micro-injection, macro-injection and micro-encapsulation techniques.
Genetic engineering does not normally include traditional animal and plant breeding, in vitro fertilisation, induction of polyploidy, mutagenesis and cell fusion techniques that do not use recombinant nucleic acids or a genetically modified organism in the process.However the European Commission has also defined genetic engineering broadly as including selective breeding and other means of artificial selection.Cloning and stem cell research, although not considered genetic engineering,are closely related and genetic engineering can be used within them.Synthetic biology is an emerging discipline that takes genetic engineering a step further by introducing artificially synthesized genetic material from raw materials into an organism.
If genetic material from another species is added to the host, the resulting organism is called transgenic. If genetic material from the same species or a species that can naturally breed with the host is used the resulting organism is called cisgenic.Genetic engineering can also be used to remove genetic material from the target organism, creating a gene knockout organism.In Europe genetic modification is synonymous with genetic engineering while within the United States of America it can also refer to conventional breeding methods.The Canadian regulatory system is based on whether a product has novel features regardless of method of origin. In other words, a product is regulated as genetically modified if it carries some trait not previously found in the species whether it was generated using traditional breeding methods (e.g., selective breeding, cell fusion, mutation breeding) or genetic engineering.Within the scientific community, the term genetic engineering is not commonly used; more specific terms such as transgenic are preferred.
Genetically modified organisms
Plants, animals or micro organisms that have changed through genetic engineering are termed genetically modified organisms or GMOs.Bacteria were the first organisms to be genetically modified. Plasmid DNA containing new genes can be inserted into the bacterial cell and the bacteria will then express those genes.These genes can code for medicines or enzymes that process food and other substrates.Plants have been modified for insect protection, herbicide resistance, virus resistance, enhanced nutrition, tolerance to environmental pressures and the production of edible vaccines.Most commercialised GMO's are insect resistant and/or herbicide tolerant crop plants.Genetically modified animals have been used for research, model animals and the production of agricultural or pharmaceutical products. They include animals with genes knocked out, increased susceptibility to disease, hormones for extra growth and the ability to express proteins in their milk.
A genetically modified organism (GMO) is an organism whose genetic material has been altered using genetic engineering techniques.Organisms that have been genetically modified include micro-organisms such as bacteria and yeast, insects, plants, fish, and mammals. GMOs are the source of genetically modified foods, and are also widely used in scientific research and to produce goods other than food. 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").
This article focuses on what organisms have been genetically engineered, and for what purposes. The article on genetic engineering focuses on the history and methods of genetic engineering, and on applications of genetic engineering and of GMOs. Both articles cover much of the same ground but with different organizations (sorted by organism in this article; sorted by application in the other). There are separate articles on genetically modified crops, genetically modified food, regulation of the release of genetic modified organisms, and controversies.
Genetic modification involves the mutation, insertion, or deletion of genes. When genes are inserted, they usually come from a different species, which is a form of horizontal gene transfer. In nature this can occur when exogenous DNA penetrates the cell membrane for any reason. To do this artificially may require attaching the genes to a virus or just physically inserting the extra DNA into the nucleus of the intended host with a very small syringe, or with very small particles fired from a gene gun.However, other methods exploit natural forms of gene transfer, such as the ability of Agrobacterium to transfer genetic material to plants,or the ability of lentiviruses to transfer genes to animal cells.
History
The general principle of producing a GMO is to alter the genetic material of an organism's genome. This may involve mutating, deleting, or adding genetic material. When genetic material from a different species is added, the resulting DNA is called recombinant DNA and the organism is called a transgenic organism. The first recombinant DNA molecules were produced by Paul Berg in 1972.
Uses
GMOs are used in biological and medical research, production of pharmaceutical drugs, experimental medicine (e.g. gene therapy), and agriculture (e.g. golden rice, resistance to herbicides). The term "genetically modified organism" does not always imply, but can include, targeted insertions of genes from one species into another. For example, a gene from a jellyfish, encoding a fluorescent protein called GFP, can be physically linked and thus co-expressed with mammalian genes to identify the location of the protein encoded by the GFP-tagged gene in the mammalian cell. Such methods are useful tools for biologists in many areas of research, including those who study the mechanisms of human and other diseases or fundamental biological processes in eukaryotic or prokaryotic cells.
Plants
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.
After thirteen years of collaborative research by 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 worldwide.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.
GM crops
In agriculture, genetically engineered crops are created to possess several desirable traits, such as resistance to pests, herbicides, or harsh environmental conditions, improved product shelf life, increased nutritional value, or production of valuable goods such as drugs (pharming). Since the first commercial cultivation of genetically modified plants in 1996, they have been modified to be tolerant to the herbicides glufosinate and glyphosate, to be resistant to virus damage as in Ringspot virus-resistant GM papaya, grown in Hawaii, and to produce the Bt toxin, an insecticide that is documented as non-toxic to mammals.
Plants, including algae, jatropha, maize, poplars and other plants have been genetically modified for use in producing fuel, known as biofuel.
Critics have objected to GM crops per se on several grounds, including ecological concerns, and economic concerns raised by the fact these organisms are subject to intellectual property law. GM crops also are involved in controversies over GM food with respect to whether food produced from GM crops is safe and whether GM crops are needed to address the world's food needs. See the genetically modified food controversies article for discussion of issues about GM crops and GM food.
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.
Microbes
Bacteria were the first organisms to be modified in the laboratory, due to their simple genetics.These organisms are now used for several purposes, and are particularly important in producing large amounts of pure human proteins for use in medicine.
Genetically modified bacteria are used to produce the protein insulin to treat diabetes.Similar bacteria have been used to produce biofuels,clotting factors to treat haemophilia,and human growth hormone to treat various forms of dwarfism.
In addition, various genetically engineered micro-organisms are routinely used as sources of enzymes for the manufacture of a variety of processed foods. These include alpha-amylase from bacteria, which converts starch to simple sugars, chymosin from bacteria or fungi that clots milk protein for cheese making, and pectinesterase from fungi which improves fruit juice clarity.
Mammals
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:
- to research human diseases (for example, to develop animal models for these diseases);
- to produce industrial or consumer products (fibres for multiple uses);
- to produce products intended for human therapeutic use (pharmaceutical products or tissue for implantation);
- to enrich or enhance the animals' interactions with humans (hypo-allergenic pets);
- to enhance production or food quality traits (faster growing fish, pigs that digest food more efficiently);
- to improve animal health (disease resistance).
Research use
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) for medical research purposes (Chalfie, Shimoura, and Tsien were awarded the Nobel prize in 2008 for GFP). For example fluorescent pigs have been bred in the US in 2000,in Korea in 2002,in Taiwan in 2006,in China in 2008 and Japan in 2009.These pigs were bred to study human organ transplants,regenerating ocular photoreceptor cells,neuronal cells in the brain,regenerative medicine via stem cells,tissue engineering,and other diseases. In 2011 a Japanese-American Team created green-fluorescent cats in order 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.
Producing human therapeutics
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.
Production or food quality traits
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 for human beings to produce milk that would be the same as human breast milk.[56] 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.
In 2006, a pig was engineered to produce omega-3 fatty acids through the expression of a roundworm gene.
Goats have been genetically engineered to produce milk with strong spiderweb-like silk proteins in their milk.
Genetically modified fish have been developed with promoters driving an over-production of growth hormone for use in the aquaculture industry to increase the speed of development and potentially reduce fishing pressure on wild stocks. AquaBounty, a biotechnology company working on bringing a GM salmon to market, claims that their GM AquAdvantage salmon can mature in half the time it takes non-GM salmon and achieves twice the size.AquaBounty has applied for regulatory approval to market their GM salmon in the US. As of May 2012 the application was still pending.
Human gene therapy
Gene therapy,uses genetically modified viruses to deliver genes that 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.Current gene therapy technology only targets the non-reproductive cells meaning that any changes introduced by the treatment can not be transmitted to the next generation. Gene therapy targeting the reproductive cells—so-called "Germ line Gene Therapy"—is very controversial and is unlikely to be developed in the near future.
Insects
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 in order to combat the spread of Dengue fever.Aedes aegypti mosquitoes, the single most important carrier of dengue fever, were reduced by 80% in a 2010 trial of these GM mosquitoes in the Cayman Islands.Between 50 and 100 million people are affected by Dengue fever every year and 40,000 people die from it.
Bollworms
A strain of Pectinophora gossypiella (Pink bollworm) has been developed that contains a fluorescent marker in their DNA. This allows researchers to monitor bollworms that have been sterilized by radiation and released in order to reduce bollworm infestation.
Aquatic life
Cnidarians
Cnidarians such as Hydra and the sea anemone Nematostella vectensis have become 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 stably transgenic hydras and sea anemones by embryo microinjection.
Fish
GM fish are used for scientific research and as pets, and are being considered for use as food and as aquatic pollution sensors.
Genetically engineered 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 (shells), 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.
Genetically modified 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, a biotechnology company working on bringing a GM salmon to market, claims that their GM AquAdvantage salmon can mature in half the time it takes non-GM salmon and achieves twice the size.AquaBounty has applied for regulatory approval to market their GM salmon in the US. As of December 2012 the application was still pending.
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.
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 coexistence of GM and nonGM crops. Depending on the coexistence regulations incentives for cultivation of GM crops differ.
Controversy
There are controversies around GMOs on several levels, including whether making them is ethical, whether food produced with them is safe, whether such food should be labeled and if so how, whether agricultural biotech is needed to address world hunger now or in the future, and more specifically to GM crops—intellectual property and market dynamics; environmental effects of GM crops; and GM crops' role in industrial agricultural more generally.
Recognition of originators
On June 19, 2013 the leaders of the three research teams who originated the technology, Robert T. Fraley of Monsanto; Marc Van Montagu of Ghent University in Belgium and founder of Plant Genetic Systems and Crop Design; and Mary-Dell Chilton of the University of Washington and Washington University in St. Louis and Syngenta were awarded with the World Food Prize. The prize, of $250,000, is awarded to people who improve the “quality, quantity or availability” of food in the world. The three competing teams first presented their results in January 1983.
History
Humans have altered the genomes of species for thousands of years through artificial selection and more recently mutagenesis. Genetic engineering as the direct manipulation of DNA by humans outside breeding and mutations has only existed since the 1970s. The term "genetic engineering" was first coined by Jack Williamson in his science fiction novel Dragon's Island, published in 1951,one year before DNA's role in heredity was confirmed by Alfred Hershey and Martha Chase,and two years before James Watson and Francis Crick showed that the DNA molecule has a double-helix structure.
In 1972 Paul Berg created the first recombinant DNA molecules by combining DNA from the monkey virus SV40 with that of the lambda virus.In 1973 Herbert Boyer and Stanley Cohen created the first transgenic organism by inserting antibiotic resistance genes into the plasmid of an E. coli bacterium.A year later Rudolf Jaenisch created a transgenic mouse by introducing foreign DNA into its embryo, making it the world’s first transgenic animal.These achievements led to concerns in the scientific community about potential risks from genetic engineering, which were first discussed in depth at the Asilomar Conference in 1975. One of the main recommendations from this meeting was that government oversight of recombinant DNA research should be established until the technology was deemed safe.
In 1976 Genentech, the first genetic engineering company was founded by Herbert Boyer and Robert Swanson and a year later the company produced a human protein (somatostatin) in E.coli. Genentech announced the production of genetically engineered human insulin in 1978.In 1980,the U.S. Supreme Court in the Diamond v.Chakrabarty case ruled that genetically altered life could be patented.The insulin produced by bacteria, branded humulin, was approved for release by the Food and Drug Administration in 1982.
In the 1970s graduate student Steven Lindow of the University of Wisconsin–Madison with D.C. Arny and C. Upper found a bacterium he identified as P. syringae that played a role in ice nucleation and in 1977, he discovered a mutant ice-minus strain. Later, he successfully created a recombinant ice-minus strain.In 1983, a biotech company, Advanced Genetic Sciences (AGS) applied for U.S. government authorization to perform field tests with the ice-minus strain of P. syringae to protect crops from frost, but environmental groups and protestors delayed the field tests for four years with legal challenges.In 1987, the ice-minus strain of P. syringae became the first genetically modified organism (GMO) to be released into the environment when a strawberry field and a potato field in California were sprayed with it.Both test fields were attacked by activist groups the night before the tests occurred: "The world's first trial site attracted the world's first field trasher".
The first field trials of genetically engineered plants occurred in France and the USA in 1986, tobacco plants were engineered to be resistant to herbicides.The People’s Republic of 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, a tomato engineered to have a longer shelf life.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.In 1995, Bt Potato was approved safe by the Environmental Protection Agency, after having been approved by the FDA, making it the first pesticide producing crop to be approved in the USA.In 2009 11 transgenic crops were grown commercially in 25 countries, the largest of which by area grown were the USA, Brazil, Argentina, India, Canada, China, Paraguay and South Africa.
In the late 1980s and early 1990s, guidance on assessing the safety of genetically engineered plants and food emerged from organizations including the FAO and WHO.
In 2010, scientists at the J. Craig Venter Institute, announced that they had created the first synthetic bacterial genome, and added it to a cell containing no DNA. The resulting bacterium, named Synthia, was the world's first synthetic life form.
Process
The first step is to choose and isolate the gene that will be inserted into the genetically modified organism. As of 2012, most commercialised GM plants have genes transferred into them that provide protection against insects or tolerance to herbicides.The gene can be isolated using restriction enzymes to cut DNA into fragments and gel electrophoresis to separate them out according to length.Polymerase chain reaction (PCR) can also be used to amplify up a gene segment, which can then be isolated through gel electrophoresis.If the chosen gene or the donor organism's genome has been well studied it may be present in a genetic library. If the DNA sequence is known, but no copies of the gene are available, it can be artificially synthesized.
The gene to be inserted into the genetically modified organism must be combined with other genetic elements in order for it to work properly. The gene can also be modified at this stage for better expression or effectiveness. As well as the gene to be inserted most constructs contain a promoter and terminator region as well as a selectable marker gene.The promoter region initiates transcription of the gene and can be used to control the location and level of gene expression, while the terminator region ends transcription. The selectable marker, which in most cases confers antibiotic resistance to the organism it is expressed in, is needed to determine which cells are transformed with the new gene. The constructs are made using recombinant DNA techniques, such as restriction digests, ligations and molecular cloning.The manipulation of the DNA generally occurs within a plasmid.
The most common form of genetic engineering involves inserting new genetic material randomly within the host genome. Other techniques allow new genetic material to be inserted at a specific location in the host genome or generate mutations at desired genomic loci capable of knocking out endogenous genes. The technique of gene targeting uses homologous recombination to target desired changes to a specific endogenous gene. This tends to occur at a relatively low frequency in plants and animals and generally requires the use of selectable markers. The frequency of gene targeting can be greatly enhanced with the use of engineered nucleases such as zinc finger nucleases,engineered homing endonucleases,or nucleases created from TAL effectors.In addition to enhancing gene targeting, engineered nucleases can also be used to introduce mutations at endogenous genes that generate a gene knockout.
Transformation
In molecular biology, transformation is genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material (exogenous DNA) from its surroundings and taken up through the cell membrane(s).Transformation occurs naturally in some species of bacteria, but it can also be effected by artificial means in other cells. For transformation to happen, bacteria must be in a state of competence, which might occur as a time-limited response to environmental conditions such as starvation and cell density.
Transformation is one of three processes by which exogenous genetic material may be introduced into a bacterial cell, the other two being conjugation (transfer of genetic material between two bacterial cells in direct contact) and transduction (injection of foreign DNA by a bacteriophage virus into the host bacterium).
"Transformation" may also be used to describe the insertion of new genetic material into nonbacterial cells, including animal and plant cells; however, because "transformation" has a special meaning in relation to animal cells, indicating progression to a cancerous state, the term should be avoided for animal cells when describing introduction of exogenous genetic material. Introduction of foreign DNA into eukaryotic cells is often called "transfection".
About 1% of bacteria are naturally able to take up foreign DNA but it can also be induced in other bacteria.Stressing the bacteria for example, with a heat shock or an electric shock, can make the cell membrane permeable to DNA that may then incorporate into their genome or exist as extrachromosomal DNA. DNA is generally inserted into animal cells using microinjection, where it can be injected through the cells nuclear envelope directly into the nucleus or through the use of viral vectors. In plants the DNA is generally inserted using Agrobacterium-mediated recombination or biolistics.
In Agrobacterium-mediated recombination the plasmid construct contains T-DNA, DNA which is responsible for insertion of the DNA into the host plants genome. This plasmid is transformed into Agrobacterium that contains no plasmids and then plant cells are infected. The Agrobacterium will then naturally insert the genetic material into the plant cells.In biolistics transformation particles of gold or tungsten are coated with DNA and then shot into young plant cells or plant embryos. Some genetic material will enter the cells and transform them. This method can be used on plants that are not susceptible to Agrobacterium infection and also allows transformation of plant plastids. Another transformation method for plant and animal cells is electroporation. Electroporation involves subjecting the plant or animal cell to an electric shock, which can make the cell membrane permeable to plasmid DNA. In some cases the electroporated cells will incorporate the DNA into their genome. Due to the damage caused to the cells and DNA the transformation efficiency of biolistics and electroporation is lower than agrobacterial mediated transformation and microinjection.
As often only a single cell is transformed with genetic material the organism must be regenerated from that single cell. As bacteria consist of a single cell and reproduce clonally regeneration is not necessary. In plants this is accomplished through the use of tissue culture. Each plant species has different requirements for successful regeneration through tissue culture. If successful an adult plant is produced that contains the transgene in every cell. In animals it is necessary to ensure that the inserted DNA is present in the embryonic stem cells. Selectable markers are used to easily differentiate transformed from untransformed cells. These markers are usually present in the transgenic organism, although a number of strategies have been developed that can remove the selectable marker from the mature transgenic plant.When the offspring is produced they can be screened for the presence of the gene. All offspring from the first generation will be heterozygous for the inserted gene and must be mated together to produce a homozygous animal.
Further testing uses PCR, Southern hybridization, and DNA sequencing is conducted to confirm that an organism contains the new gene. These tests can also confirm the chromosomal location and copy number of the inserted gene. The presence of the gene does not guarantee it will be expressed at appropriate levels in the target tissue so methods that look for and measure the gene products (RNA and protein) are also used. These include northern hybridization, quantitative RT-PCR, Western blot, immunofluorescence, ELISA and phenotypic analysis. For stable transformation the gene should be passed to the offspring in a Mendelian inheritance pattern, so the organism's offspring are also studied.
History
Transformation was first demonstrated in 1928 by British bacteriologist Frederick Griffith. Griffith discovered that a harmless strain of Streptococcus pneumoniae could be made virulent after being exposed to heat-killed virulent strains. Griffith hypothesized that some "transforming principle" from the heat-killed strain was responsible for making the harmless strain virulent. In 1944 this "transforming principle" was identified as being genetic by Oswald Avery, Colin MacLeod, and Maclyn McCarty. They isolated DNA from a virulent strain of S. pneumoniae and using just this DNA were able to make a harmless strain virulent. They called this uptake and incorporation of DNA by bacteria "transformation" (See Avery-MacLeod-McCarty experiment). The results of Avery et al.'s experiments were at first skeptically received by the scientific community and it was not until the development of genetic markers and the discovery of other methods of genetic transfer (conjugation in 1947 and transduction in 1953) by Joshua Lederberg that Avery's experiments were accepted.
It was originally thought that Escherichia coli, a commonly used laboratory organism, was refractory to transformation. However, in 1970, Morton Mandel and Akiko Higa showed that E. coli may be induced to take up DNA from bacteriophage λ without the use of helper phage after treatment with calcium chloride solution.Two years later in 1972, Stanley Cohen, Annie Chang and Leslie Hsu showed that CaCl2 treatment is also effective for transformation of plasmid DNA.The method of transformation by Mandel and Higa was later improved upon by Douglas Hanahan.The discovery of artificially induced competence in E. coli created an efficient and convenient procedure for transforming bacteria which allows for simpler molecular cloning methods in biotechnology and research, and it is now a routinely used laboratory procedure.
Transformation using electroporation was developed in the late 1980s, increasing the efficiency of in-vitro transformation and increasing the number of bacterial strains that could be transformed.Transformation of animal and plant cells was also investigated with the first transgenic mouse being created by injecting a gene for a rat growth hormone into a mouse embryo in 1982.In 1907 a bacterium that caused plant tumors, Agrobacterium tumefaciens, was discovered and in the early 1970s the tumor inducing agent was found to be a DNA plasmid called the Ti plasmid.By removing the genes in the plasmid that caused the tumor and adding in novel genes researchers were able to infect plants with A. tumefaciens and let the bacteria insert their chosen DNA into the genomes of the plants.Not all plant cells are susceptible to infection by A. tumefaciens so other methods were developed including electroporation and micro-injection.Particle bombardment was made possible with the invention of the Biolistic Particle Delivery System (gene gun) by John Sanford in the 1980s.
Methods and mechanisms
Bacteria
Bacterial transformation may be referred to as a stable genetic change brought about by the uptake of naked DNA (DNA without associated cells or proteins) to increase DNA quantity and competence refers to the state of being able to take up exogenous DNA from the environment. There are two forms of transformation and competence: natural and artificial.
Natural transformation
Natural transformation is a bacterial adaptation for DNA transfer that depends on the expression of numerous bacterial genes whose products appear to be designed to carry out this process.In general, transformation is a complex, energy requiring developmental process. In order for a bacterium to bind, take up and recombine exogenous DNA into its chromosome it must become competent, that is, enter a special physiological state. Competence development in Bacillus subtilis requires expression of about 40 genes.The DNA integrated into the host chromosome is usually (but with rare exceptions) derived from another bacterium of the same species, and is thus homologous to the resident chromosome.
In B. subtilis the length of the transferred DNA is greater than 1271 kb (more than 1 million bases).The length transferred is likely double stranded DNA and is often more than a third of the total chromosome length of 4215 kb.It appears that about 7-9% of the recipient cells take up an entire chromosome.
The capacity for natural transformation appears to occur in a number of prokaryotes, and thus far 67 prokaryotic species (in seven different phyla) are known to undergo this process.
Competence for transformation is typically induced by high cell density and/or nutritional limitation, conditions associated with the stationary phase of bacterial growth. Transformation in Haemophilus influenzae occurs most efficiently at the end of exponential growth as bacterial growth approaches stationary phase.Transformation in Streptococcus mutans, as well as in many other streptococci, occurs at high cell density and is associated with biofilm formation.Competence in B. subtilis is induced toward the end of logarithmic growth, especially under conditions of amino acid limitation.
Transformation, as an adaptation for DNA repair
Competence is specifically induced by DNA damaging conditions. For instance, transformation is induced in Streptococcus pneumoniae by the DNA damaging agents mitomycin C (a DNA crosslinking agent) and fluoroquinolone (a topoisomerase inhibitor that causes double-strand breaks).In B. subtilis, transformation is increased by UV light, a DNA damaging agent.In Helicobacter pylori, ciprofloxacin, which interacts with DNA gyrase and introduces double-strand breaks, induces expression of competence genes, thus enhancing the frequency of transformation Using Legionella pneumophila, Charpentier et al.tested 64 toxic molecules to determine which of these induce competence. Of these, only six, all DNA damaging agents caused strong induction. These DNA damaging agents were mitomycin C (which causes DNA inter-strand crosslinks), norfloxacin, ofloxacin and nalidixic acid (inhibitors of DNA gyrase that cause double-strand breaks),bicyclomycin (causes single- and double-strand breaks), and hydroxyurea (induces DNA base oxidation). UV light also induced competence in L. pneumophila. Charpentier et al.suggested that competence for transformation probably evolved as a DNA damage response.
Logarithmically growing bacteria differ from stationary phase bacteria with respect to the number of genome copies present in the cell, and this has implications for the capability to carry out an important DNA repair process. During logarithmic growth, two or more copies of any particular region of the chromosome may be present in a bacterial cell, as cell division is not precisely matched with chromosome replication. The process of homologous recombinational repair (HRR) is a key DNA repair process that is especially effective for repairing double-strand damages, such as double-strand breaks. This process depends on a second homologous chromosome in addition to the damaged chromosome. During logarithmic growth, a DNA damage in one chromosome may be repaired by HRR using sequence information from the other homologous chromosome. Once cells approach stationary phase, however, they typically have just one copy of the chromosome, and HRR requires input of homologous template from outside the cell by transformation.
To test whether the adaptive function of transformation is repair of DNA damages, a series of experiments were carried out using B. subtilis irradiated by UV light as the damaging agent (reviewed by Michod et al.and Bernstein et al.The results of these experiments indicated that transforming DNA acts to repair potentially lethal DNA damages introduced by UV light in the recipient DNA. The particular process responsible for repair was likely HRR. Transformation in bacteria can be viewed as a primitive sexual process, since it involves interaction of homologous DNA from two individuals to form recombinant DNA that is passed on to succeeding generations. Bacterial transformation in prokaryotes may have been the ancestral process that gave rise to meiotic sexual reproduction in eukaryotes.
Natural competence
About 1% of bacterial species are capable of naturally taking up DNA under laboratory conditions; more may be able to take it up in their natural environments. DNA material can be transferred between different strains of bacteria, in a process that is called horizontal gene transfer. Some species upon cell death release their DNA to be taken up by other cells, however transformation works best with DNA from closely related species. These naturally competent bacteria carry sets of genes that provide the protein machinery to bring DNA across the cell membrane(s).The transport of the exogeneous DNA into the cells may require proteins that are involved in the assembly of type IV pili and type II secretion system, as well as DNA translocase complex at the cytoplasmic membrane.
Due to the differences in structure of the cell envelope between Gram-positive and Gram-negative bacteria, there are some differences in the mechanisms of DNA uptake in these cells, however most of them share common features that involve related proteins. The DNA first binds to the surface of the competent cells on a DNA receptor, and passes through the cytoplasmic membrane via DNA translocase.Only single-stranded DNA may pass through, one strand is therefore degraded by nucleases in the process, and the translocated single-stranded DNA may then be integrated into the bacterial chromosomes by a RecA-dependent process. In Gram-negative cells, due to the presence of an extra membrane, the DNA requires the presence of a channel formed by secretins on the outer membrane. Pilin may be required for competence however its role is uncertain.The uptake of DNA is generally non-sequence specific, although in some species the presence of specific DNA uptake sequences may facilitate efficient DNA uptake.
Artificial competence
Schematic of bacterial transformation — for which artificial competence must first be induced.
Artificial competence can be induced in laboratory procedures that involve making the cell passively permeable to DNA by exposing it to conditions that do not normally occur in nature.Typically the cells are incubated in a solution containing divalent cations (often calcium chloride) under cold conditions, before being exposed to a heat pulse (heat shock).The mechanism of the uptake of DNA via chemically induced competence in this calcium chloride transformation method has for a long time been unclear.
It has been found that growth of Gram negative bacteria in 20 mM Mg reduces the number of protein to lipopolysaccharide bonds by increasing the ratio of ionic to covalent bonds, which increases membrane fluidity, facilitating transformation. The role of lipopolysaccharides here are verified from the observation that shorter O-side chains are more effectively transformed — perhaps because of improved DNA accessibility.
The surface of bacteria such as E. coli is negatively charged due to phospholipids and lipopolysaccharides on its cell surface, and the DNA is also negatively charged. One function of the divalent cation therefore would be to shield the charges by coordinating the phosphate groups and other negative charges, thereby allowing a DNA molecule to adhere to the cell surface.
DNA entry into E. coli cells is through channels known as zones of adhesion or Bayer’s junction, a typical cell carrying as many as 400 such zones. Their role was established when cobalamine (which also uses these channels) was found to competitively inhibit DNA uptake. Another type of channel implicated in DNA uptake consists of poly (HB):poly P:Ca. In this poly (HB) is envisioned to wrap around DNA (itself a polyphosphate), and is carried in a shield formed by Ca ions.
It is suggested that exposing the cells to divalent cations in cold condition may also change or weaken the cell surface structure of the cells making it more permeable to DNA. The heat-pulse is thought to create a thermal imbalance on either side of the cell membrane, which forces the DNA to enter the cells through either cell pores or the damaged cell wall.
Electroporation is another method of promoting competence. In this method the cells are briefly shocked with an electric field of 10-20 kV/cm which is thought to create holes in the cell membrane through which the plasmid DNA may enter. After the electric shock the holes are rapidly closed by the cell's membrane-repair mechanisms.
Yeast
Most species of yeast, including Saccharomyces cerevisiae, may be transformed by exogenous DNA in the environment. Several methods have been developed to facilitate this transformation at high frequency in the lab.
Yeast cells may be treated with enzymes to degrade their cell walls, yielding spheroplasts. These cells are very fragile but take up foreign DNA at a high rate.
Exposing intact yeast cells to alkali cations such as those of cesium or lithium allows the cells to take up plasmid DNA.Later protocols adapted this transformation method, using lithium acetate, polyethylene glycol, and single-stranded DNA.In these protocols, the single-stranded DNA preferentially binds to the yeast cell all, preventing plasmid DNA from doing so and leaving it available for transformation.
Electroporation : Formation of transient holes in the cell membranes using electric shock; this allow DNA to enter as described above for Bacteria.
Enzymatic digestion or agitation with glass beads may also be used to transform yeast cells.
Plants
Agrobacterium mediated transformation is the easiest and most simple plant transformation. Plant tissue (often leaves) are cut into small pieces, e.g. 10x10mm, and soaked for 10 minutes in a fluid containing suspended Agrobacterium. Some cells along the cut will be transformed by the bacterium, that inserts its DNA into the cell. Placed on selectable rooting and shooting media, the plants will regrow. Some plants species can be transformed just by dipping the flowers into suspension of Agrobacterium and then planting the seeds in a selective medium. Unfortunately, many plants are not transformable by this method.
Gene gun : Also referred to as particle bombardment, microprojectile bombardment, or biolistics. Particles of gold or tungsten are coated with DNA and then shot into young plant cells or plant embryos. Some genetic material will stay in the cells and transform them. This method also allows transformation of plant plastids. The transformation efficiency is lower than in Agrobacterium mediated transformation, but most plants can be transformed with this method.
Electroporation : Formation of transient holes in cell membranes using electric shock; this allows DNA to enter as described above for Bacteria.
Viral transformation (transduction) : Package the desired genetic material into a suitable plant virus and allow this modified virus to infect the plant. If the genetic material is DNA, it can recombine with the chromosomes to produce transformant cells. However genomes of most plant viruses consist of single stranded RNA which replicates in the cytoplasm of infected cell. For such genomes this method is a form of transfection and not a real transformation, since the inserted genes never reach the nucleus of the cell and do not integrate into the host genome. The progeny of the infected plants is virus free and also free of the inserted gene.
Animals
Introduction of DNA into animal cells is usually called transfection,and is discussed in the corresponding article.
Transfection is the process of deliberately introducing nucleic acids into cells.The term is often used for non-viral methods in eukaryotic cells.It may also refer to other methods and cell types, although other terms are preferred: "transformation" is more often used to describe non-viral DNA transfer in bacteria, non-animal eukaryotic cells and plant cells – a distinctive sense of transformation refers to spontaneous genetic modifications (mutations to cancerous cells (carcinogenesis), or under stress (UV irradiation)). Transduction is often used to describe virus-mediated DNA transfer. The word transfection is a blend of trans- and infection.
Genetic material (such as supercoiled plasmid DNA or siRNA constructs), or even proteins such as antibodies, may be transfected.
Transfection of animal cells typically involves opening transient pores or "holes" in the cell membrane, to allow the uptake of material. Transfection can be carried out using calcium phosphate, by electroporation, or by mixing a cationic lipid with the material to produce liposomes, which fuse with the cell membrane and deposit their cargo inside.
Transfection can result in unexpected morphologies and abnormalities in target cells.
Practical aspects of transformation in molecular biology
The discovery of artificially induced competence in bacteria allow bacteria such as Escherichia coli to be used as a convenient host for the manipulation of DNA as well as expressing proteins. Typically plasmids are used for transformation in E. coli. In order to be stably maintained in the cell, a plasmid DNA molecule must contain an origin of replication, which allows it to be replicated in the cell independently of the replication of the cell's own chromosome.
The efficiency with which a competent culture can take up exogenous DNA and express its genes is known as Transformation efficiency and is measured in colony forming unit (cfu) per μg DNA used. A transformation efficiency of 1x108 cfu/μg for a small plasmid like pUC19 is roughly equivalent to 1 in 2000 molecules of the plasmid used being transformed.
In calcium chloride transformation, the cells are prepared by chilling cells in the presence of Ca2+ (in CaCl2 solution) making the cell become permeable to plasmid DNA. The cells are incubated on ice with the DNA, and then briefly heat-shocked (e.g., at 42°C for 30–120 seconds). This method works very well for circular plasmid DNA. Non-commercial preparations should normally give 106 to 107 transformants per microgram of plasmid; a poor preparation will be about 104/μg or less, but a good preparation of competent cells can give up to ~108 colonies per microgram of plasmid.Protocols however exist for making supercompetent cells that may yield a transformation efficiency of over 109.The chemical method, however, usually does not work well for linear DNA, such as fragments of chromosomal DNA, probably because the cell's native exonuclease enzymes rapidly degrade linear DNA. In contrast, cells that are naturally competent are usually transformed more efficiently with linear DNA than with plasmid DNA.
The transformation efficiency using the CaCl2 method decreases with plasmid size, and electroporation therefore may be a more effective method for the uptake of large plasmid DNA.Cells used in electroporation should be prepared first by washing in cold double-distilled water to remove charged particles that may create sparks during the electroporation process.
Selection and screening in plasmid transformation
Because transformation usually produces a mixture of relatively few transformed cells and an abundance of non-transformed cells, a method is necessary to select for the cells that have acquired the plasmid. The plasmid therefore requires a selectable marker such that those cells without the plasmid may be killed or have their growth arrested. Antibiotic resistance is the most commonly used marker for prokaryotes. The transforming plasmid contains a gene that confers resistance to an antibiotic that the bacteria are otherwise sensitive to. The mixture of treated cells is cultured on media that contain the antibiotic so that only transformed cells are able to grow. Another method of selection is the use of certain auxotrophic markers that can compensate for an inability to metabolise certain amino acids, nucleotides, or sugars. This method requires the use of suitably mutated strains that are deficient in the synthesis or utility of a particular biomolecule, and the transformed cells are cultured in a medium that allows only cells containing the plasmid to grow.
In a cloning experiment, a gene may be inserted into a plasmid used for transformation. However, in such experiment, not all the plasmids may contain a successfully inserted gene. Additional techniques may therefore be employed further to screen for transformed cells that contain plasmid with the insert. Reporter genes can be used as markers, such as the lacZ gene which codes for β-galactosidase used in blue-white screening. This method of screening relies on the principle of α-complementation, where a fragment of the lacZ gene (lacZα) in the plasmid can complement another mutant lacZ gene (lacZΔM15) in the cell. Both genes by themselves produce non-functional peptides, however, when expressed together, as when a plasmid containing lacZ-α is transformed into a lacZΔM15 cells, they form a functional β-galactosidase. The presence of an active β-galactosidase may be detected when cells are grown in plates containing X-gal, forming characteristic blue colonies. However, the multiple cloning site, where a gene of interest may be ligated into the plasmid vector, is located within the lacZα gene. Successful ligation therefore disrupts the lacZα gene, and no functional β-galactosidase can form, resulting in white colonies. Cells containing successfully ligated insert can then be easily identified by its white coloration from the unsuccessful blue ones.
Other commonly used reporter genes are green fluorescent protein (GFP), which produces cells that glow green under blue light, and the enzyme luciferase, which catalyzes a reaction with luciferin to emit light. The recombinant DNA may also be detected using other methods such as nucleic acid hybridization with radioactive RNA probe, while cells that expressed the desired protein from the plasmid may also be detected using immunological methods.
Genome editing
Genome editing is a type of genetic engineering in which DNA is inserted, replaced, or removed from a genome using artificially engineered nucleases, or "molecular scissors." The nucleases create specific double-stranded break (DSBs) at desired locations in the genome, and harness the cell’s endogenous mechanisms to repair the induced break by natural processes of homologous recombination (HR) and nonhomologous end-joining (NHEJ). There are currently four families of engineered nucleases: meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPRs.
Applications
Genetic engineering has applications in medicine, research, industry and agriculture and can be used on a wide range of plants, animals and micro organisms.
Medicine
In medicine genetic engineering has been used to mass-produce insulin, human growth hormones, follistim (for treating infertility), human albumin, monoclonal antibodies, antihemophilic factors, vaccines and many other drugs.Vaccination generally involves injecting weak live, killed or inactivated forms of viruses or their toxins into the person being immunized.Genetically engineered viruses are being developed that can still confer immunity, but lack the infectious sequences. Mouse hybridomas, cells fused together to create monoclonal antibodies, have been humanised through genetic engineering to create human monoclonal antibodies.Genetic engineering has shown promise for treating certain forms of cancer.
Genetic engineering is used to create animal models of human diseases. Genetically modified mice are the most common genetically engineered animal model.They have been used to study and model cancer (the oncomouse), obesity, heart disease, diabetes, arthritis, substance abuse, anxiety, aging and Parkinson disease.Potential cures can be tested against these mouse models. Also genetically modified pigs have been bred with the aim of increasing the success of pig to human organ transplantation.
Gene therapy is the genetic engineering of humans by replacing defective human genes with functional copies. This can occur in somatic tissue or germline tissue. If the gene is inserted into the germline tissue it can be passed down to that person's descendants.Gene therapy has been successfully used to treat multiple diseases, including X-linked SCID, gene therapy treatment to be approved for clinical use in either Europe or the United States after its endorsement by the European Commission.There are also ethical concerns should the technology be used not just for treatment, but for enhancement, modification or alteration of a human beings' appearance, adaptability, intelligence, character or behavior.The distinction between cure and enhancement can also be difficult to establish.Transhumanists consider the enhancement of humans desirable.
Research
Human cells in which some proteins are fused with green fluorescent protein to allow them to be visualised
Genetic engineering is an important tool for natural scientists. Genes and other genetic information from a wide range of organisms are transformed into bacteria for storage and modification, creating genetically modified bacteria in the process. Bacteria are cheap, easy to grow, clonal, multiply quickly, 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.
Organisms are genetically engineered to discover the functions of certain genes. This could be the effect on the phenotype of the organism, where the gene is expressed or what other genes it interacts with. These experiments generally involve loss of function, gain of function, tracking and expression.
Loss of function experiments, such as in a gene knockout experiment, in which an organism is engineered to lack the activity of one or more genes. A knockout experiment involves the creation and manipulation of a DNA construct in vitro, which, in a simple knockout, consists of a copy of the desired gene, which has been altered such that it is non-functional. Embryonic stem cells incorporate the altered gene, which replaces the already present functional copy. These stem cells are injected into blastocysts, which are implanted into surrogate mothers. This allows the experimenter to analyze the defects caused by this mutation and thereby determine the role of particular genes. It is used especially frequently in developmental biology. Another method, useful in organisms such as Drosophila (fruit fly), is to induce mutations in a large population and then screen the progeny for the desired mutation. A similar process can be used in both plants and prokaryotes.
Gain of function experiments, the logical counterpart of knockouts. These are sometimes performed in conjunction with knockout experiments to more finely establish the function of the desired gene. The process is much the same as that in knockout engineering, except that the construct is designed to increase the function of the gene, usually by providing extra copies of the gene or inducing synthesis of the protein more frequently.
Tracking experiments, which seek to gain information about the localization and interaction of the desired protein. One way to do this is to replace the wild-type gene with a 'fusion' gene, which is a juxtaposition of the wild-type gene with a reporting element such as green fluorescent protein (GFP) that will allow easy visualization of the products of the genetic modification. While this is a useful technique, the manipulation can destroy the function of the gene, creating secondary effects and possibly calling into question the results of the experiment. More sophisticated techniques are now in development that can track protein products without mitigating their function, such as the addition of small sequences that will serve as binding motifs to monoclonal antibodies.
Expression studies aim to discover where and when specific proteins are produced. In these experiments, the DNA sequence before the DNA that codes for a protein, known as a gene's promoter, is reintroduced into an organism with the protein coding region replaced by a reporter gene such as GFP or an enzyme that catalyzes the production of a dye. Thus the time and place where a particular protein is produced can be observed. Expression studies can be taken a step further by altering the promoter to find which pieces are crucial for the proper expression of the gene and are actually bound by transcription factor proteins; this process is known as promoter bashing.
Industrial
Using genetic engineering techniques one can transform microorganisms such as bacteria or yeast, or insect mammalian cells with a gene coding for a useful protein, such as an enzyme, so that the transformed organism will overexpress the desired protein. One can manufacture mass quantities of the protein by growing the transformed organism in bioreactor equipment using techniques of industrial fermentation, and then purifying the protein.Some genes do not work well in bacteria, so yeast, insect cells, or mammalians cells, each a eukaryote, can also be used.These techniques are used to produce medicines such as insulin, human growth hormone, and vaccines, supplements such as tryptophan, aid in the production of food (chymosin in cheese making) and fuels.Other applications involving genetically engineered bacteria being investigated involve making the bacteria perform tasks outside their natural cycle, such as making biofuels,cleaning up oil spills, carbon and other toxic waste and detecting arsenic in drinking water.
Experimental, lab scale industrial applications
In materials science, a genetically modified virus has been used in an academic lab as a scaffold for assembling a more environmentally friendly lithium-ion battery.
Bacteria have been engineered to function as sensors by expressing a fluorescent protein under certain environmental conditions.
Agriculture
One of the best-known and controversial applications of genetic engineering is the creation and use of genetically modified crops or genetically modified organisms, such as genetically modified fish, which are used to produce genetically modified food and materials with diverse uses. There are four main goals in generating genetically modified crops.
One goal, and the first to be realized commercially, is to provide protection from environmental threats, such as cold (in the case of Ice-minus bacteria), or pathogens, such as insects or viruses, and/or resistance to herbicides. There are also fungal and virus resistant crops developed or in development.They have been developed to make the insect and weed management of crops easier and can indirectly increase crop yield.
Another goal in generating GMOs, is to modify the quality of the produce, for instance, increasing the nutritional value or providing more industrially useful qualities or quantities of the produce.The Amflora potato, for example, produces a more industrially useful blend of starches. Cows have been engineered to produce more protein in their milk to facilitate cheese production.Soybeans and canola have been genetically modified to produce more healthy oils.
Another goal consists of driving the GMO to produce materials that it does not normally make. One example is "pharming", which uses crops as bioreactors to produce vaccines, drug intermediates, or drug themselves; the useful product is purified from the harvest and then used in the standard pharmaceutical production process.Cows and goats have been engineered to express drugs and other proteins in their milk, and in 2009 the FDA approved a drug produced in goat milk.
Another goal in generating GMOs, is to directly improve yield by accelerating growth, or making the organism more hardy (for plants, by improving salt, cold or drought tolerance).Some agriculturally important animals have been genetically modified with growth hormones to increase their size.
The genetic engineering of agricultural crops can increase the growth rates and resistance to different diseases caused by pathogens and parasites.This is beneficial as it can greatly increase the production of food sources with the usage of fewer resources that would be required to host the world's growing populations. These modified crops would also reduce the usage of chemicals, such as fertilizers and pesticides, and therefore decrease the severity and frequency of the damages produced by these chemical pollution.
Ethical and safety concerns have been raised around the use of genetically modified food.A major safety concern relates to the human health implications of eating genetically modified food, in particular whether toxic or allergic reactions could occur.Gene flow into related non-transgenic crops, off target effects on beneficial organisms and the impact on biodiversity are important environmental issues.Ethical concerns involve religious issues, corporate control of the food supply, intellectual property rights and the level of labeling needed on genetically modified products.
BioArt and entertainment
Genetic engineering is also being used to create BioArt.Some bacteria have been genetically engineered to create black and white photographs
Genetic engineering has also been used to create novelty items such as lavender-colored carnations,blue roses,and glowing fish.
Regulation
The regulation of genetic engineering concerns the approaches taken by governments to assess and manage the risks associated with the development and release of genetically modified crops. There are differences in the regulation of GM crops 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.
Controversy
Critics have objected to use of genetic engineering per se on several grounds, including ethical concerns, ecological concerns, and economic concerns raised by the fact GM techniques and GM organisms are subject to intellectual property law. GMOs also are involved in controversies over GM food with respect to whether food produced from GM crops is safe, whether it should be labeled, and whether GM crops are needed to address the world's food needs. See the genetically modified food controversies article for discussion of issues about GM crops and GM food. These controversies have led to litigation, international trade disputes, and protests, and to restrictive regulation of commercial products in most countries.
The Future of Genetic Engineering
Genetic Engineering has the potential to fundamentally affect our daily lives and now it has already change the practice of medicine, the production of food, and the synthesis of drugs.
The effect of this genetic revolution so to speak will be for reaching throughout society most especially medicine, agricultural, and manufacturing.Many of the technical problems associated with early applications of genetic engineering will be overcome.
As scientists further develop the technology of genetic engineering, the application of this technology will become more luractive. Increasingly, genetic engineering will be applied in different industries and will continue to become an important factor in the global economy.
