genetic engineering

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Concept

Genetic engineering is the alteration of genetic material by direct intervention in genetic processes with the purpose of producing new substances or improving functions of existing organisms. It is a very young, exciting, and controversial branch of the biological sciences. On the one hand, it offers the possibility of cures for diseases and countless material improvements to daily life. Hopes for the benefits of genetic engineering are symbolized by the Human Genome Project, a vast international effort to categorize all the genes in the human species. On the other hand, genetic engineering frightens many with its potential for misuse, either in Nazi-style schemes for population control or through simple bungling that might produce a biological holocaust caused by a man-made virus. Symbolic of the alarming possibilities is the furor inspired by a single concept on the cutting edge of genetic engineering: cloning.

How It Works

Dna

Any discussion of genetics makes reference to DNA (deoxyribonucleic acid), a molecule that contains genetic codes for inheritance. DNA resides in chromosomes, threadlike structures found in the nucleus, or control center, of every cell in every living thing. Chromosomes themselves are made up of genes, which carry codes for the production of proteins. The latter, of which there are many thousands of different varieties, make up the majority of the human body's dry weight.

Although it is central to the latest advances in modern genetic research, DNA was discovered more than 130 years ago. In 1869 the Swiss biochemist Johann Friedrich Miescher (1844-1895) isolated a substance, containing both nitrogen and phosphorus, that separated into a protein and an acid molecule. He called it nucleic acid, and in this material he discovered DNA. Some 74 years would pass, however, before scientists recognized the function of the nucleic acid Miescher had discovered. Then, in 1944, a research team led by the Canadian-born American bacteriologist Oswald Avery (1877-1955) found that by taking DNA from one type of bacterium and inserting it into another, the second bacterium took on certain traits of the first. This experiment, along with other experiments and research, proved that DNA serves as a blueprint for the characteristics and functions of organisms.

The Double Helix

Nine years later, in 1953, the American biochemist James D. Watson (1928-) and the English biochemist Francis Crick (1916-) solved the mystery of DNA's structure and explained the means by which it provides necessary instructions at critical moments in the course of cell division and growth. They proposed a double helix, or spiral staircase, model, which linked the chemical bases of DNA in definite pairs. Using this twisted-ladder model, they were able to explain how the DNA molecule could duplicate itself, since each side of the ladder is identical to the other; if separated, each would serve as the template for the formation of its mirror image.

The sides of the DNA ladder are composed of alternating sugar and phosphate molecules, like links in a chain, and consist of four different chemical bases: adenine, guanine, cytosine, and thymine. The four letters designating these bases—A, G, C, and T—are the alphabet of the genetic code, and each rung of the DNA molecule is made up of a combination of two of these letters. Owing to specific chemical affinities, A always combines with T and C with G, to form what is called a base pair. Specific sequences of these base pairs, which are bonded to each other by atoms of hydrogen, constitute the genes.

Endless Combinations

A four-letter alphabet may seem rather small for constructing the extensive vocabulary that defines the myriad life-forms on Earth. If one stops to consider the exponential operations involved, however, it is easy to understand how large the range of possibilities can become. For any sequence, there are four possibilities for the first two letters (AT, TA, CG, or GC) and four more possibilities for the second two letters. Thus, just for a four-letter sequence, there are 16 possibilities, and for each pair of letters added to the sequence, the total is multiplied by four.

To see where this might lead, imagine that you started with a penny and tried to quadruple your funds every day. The first day there would not be a dramatic increase, since you would have to earn only $0.04, and even by day 4 you would need only $2.56 to meet your goal. But as the quadrupling process continued, day by day the sums of money would get bigger ($655.36 on day 8) and bigger ($16,772.16 on day 12) and bigger ($687,194,767.36 on day 18). Given the fact that the human body contains an almost unfathomable number of genes, each of which may be between 2,000 and 200,000 base pairs long, one can begin to imagine just how large the number of possibilities would become.

Each one of these combinations has a different meaning, providing the code for all manner of specific traits, such as brown hair and blue eyes, dimples, unattached earlobes, and so on. Except for identical twins, no two humans have exactly the same genetic information. What follows are just a few facts about the human genome—that is, all of the genetic material in the chromosomes of the human organism:

Some Facts About the Human Genome

• The human body contains about 100 trillion cells.
• Each cell has a DNA code consisting of some 1.5 billion base pairs.
• The DNA in each cell, if stretched to its full length, would be 6 ft. (1.8 m) long—yet it fits into a space about 0.0004 in. (0.0001 cm) across, smaller than the head of a pin.
• If all of the DNA in the human body were stretched end to end, it would reach to the Sun and back more than 600 times.
• If a person attempted to recite the entire human genome, with all its base pairs, at the rate of one letter per second, 24 hours a day, it would take a century.
• Every second scientists working on the Human Genome Project are decoding some 12,000 letters of DNA.
• Our DNA is 98% identical to that of chimpanzees.
• Only 0.2% of all human DNA differs between individuals; in other words, people are 99.8% the same, and all the vast differences between people are a product of just 1/500th of the total DNA.
• Despite all that scientists know about DNA, a staggering 97% of all human DNA has no known function.

Principles of Genetic Engineering

Just as DNA is at the core of studies in genetics, recombinant DNA (rDNA)—that is, DNA that has been genetically altered through a process known as gene splicing—is the focal point of genetic engineering. In gene splicing, a DNA strand is cut in half lengthwise and joined with a strand from another organism or perhaps even another species. Use of gene splicing makes possible two other highly significant techniques. Gene transfer, or incorporation of new DNA into an organism's cells, usually is carried out with the help of a microorganism that serves as a vector, or carrier. Gene therapy is the introduction of normal or genetically altered genes to cells, generally to replace defective genes involved in genetic disorders.

DNA also can be cut into shorter fragments through the use of restriction enzymes. (An enzyme is a type of protein that speeds up chemical reactions.) The ends of these fragments have an affinity for complementary ends on other DNA fragments and will seek those out in the target DNA. By looking at the size of the fragment created by a restriction enzyme, investigators can determine whether the gene has the proper genetic code. This technique has been used to analyze genetic structures in fetal cells and to diagnose certain blood disorders, such as sickle cell anemia.

Gene Transfer

Suppose that a particular base-pair sequence carries the instruction "make insulin"; if a way could be found to insert that base sequence into the DNA of bacteria, for example, those bacteria would be capable of manufacturing insulin. This, in turn, would greatly improve the lives of people with type 1 diabetes, who depend on insulin shots to aid their bodies in processing blood sugar. (See Non-infectious Diseases for more about diabetes.)

Although the concept of gene transfer is relatively simple, its execution presents considerable technical obstacles. The first person to surmount these obstacles was the American biochemist Paul Berg (1926-), often referred to as the "father of genetic engineering." In 1973 Berg developed a method for joining the DNA from two different organisms, a monkey virus known as SV40 and a virus called lambda phage. Although the accomplishment was clearly a breakthrough, Berg's method was difficult. Then, later that year, the American biochemists Stanley Cohen (1922-) at Stanford University, and Herbert Boyer (1936-) at the University of California at San Francisco discovered an enzyme that greatly increased the efficiency of the Berg procedure. The gene-transfer technique developed by Berg, Boyer, and Cohen formed the basis for much of the ensuing progress in genetic engineering.

Real-Life Applications

Big Business in Dna

Ever since the breakthrough discoveries of Watson, Crick, and others in the 1950s made genetic engineering a possibility, the new field has promised increasingly bigger payoffs. These payoffs take the form of improvements to human life and profits to those who facilitate those improvements. The possible applications of genetic engineering are virtually limitless—as are the profits to be made from genetic engineering as a business. As early as the 1970s, entrepreneurs (independent businesspeople) recognized the commercial potential of genetically engineered products, which promised to revolutionize life, technology, and commerce as computers also were doing. Thus was born one of the great buzzwords of the late twentieth century: biotechnology, or the use of genetic engineering for commercial purposes.

Several early biotechnology firms were founded by scientists involved in fundamental research: Boyer, for example, teamed up with the venture capitalist Robert Swanson in 1976 to form Genentech (Genetic Engineering Technology). Other pioneering companies, including Cetus, Biogen, and Genex, likewise were founded through the collaboration of scientists and businesspeople. Today biotechnology promises a revolution in numerous areas, such as agriculture. Recombinant DNA techniques enable scientists to produce plants that are resistant to freezing temperatures, that will take longer to ripen, that will develop their own resistance to pests, and so on. By 1988 scientists had tested more than two dozen kinds of plants engineered to have special properties such as these. Yet no field of biotechnology and genetic engineering is as significant as the applications to health and the cures for diseases.

Medicines and Cures

The use of rDNA allows scientists to produce many products that were previously available only in limited quantities: for example, insulin, which we referred to earlier. Until the 1980s the only source of insulin for people with diabetes came from animals slaughtered for meat and other purposes. The supply was never high enough to meet demand, and this drove up prices. Then, in 1982, the U.S. Food and Drug Administration (FDA) approved the sale of insulin produced by genetically altered organisms—the first such product to become available. Since 1982 several additional products, such as human growth hormone, have been made with rDNA techniques.

One of the most exciting potential applications of genetic engineering is the treatment of genetic disorders, which are discussed in Heredity, through the use of gene therapy. Among the more than 3,000 such disorders, quite a few of which are quite serious or even fatal, many are the result of relatively minor errors in DNA sequencing. Genetic engineering offers the potential to provide individuals with correct copies of a gene, which could make possible a cure for that condition. In the 1980s scientists began clinical trials of a procedure known as human gene therapy to replace defective genes. The technique, still very much in the developmental stage, offers the hope of cures for diseases that medicine has long been powerless to combat.

In 2001 scientists at the Weizmann Institute in Israel brought together two of the most exciting fields of research, biotechnology and computers, to produce the DNA-processing nanocomputer. It is an actual computer, but it is so small that a trillion of them would fit in a test tube. It consists of DNA and DNA-processing enzymes, both dissolved in liquid; thus its input, output, and software are all in the form of DNA molecules. The purpose of the nanocomputer is to analyze DNA, detecting abnormalities in the human body and creating remedies for them.

The Human Genome Project

At the center of genetic studies, with vast potential applications to genetic engineering, is the Human Genome Project (HGP), an international effort to analyze and map the DNA of humans and several other organisms. As discussed in the essay Genetics, the HGP began with efforts by the Atomic Energy Commission, a predecessor to the U.S. Department of Energy, to study the genetic effects of radioactive nuclear fallout. In 1990 the Department of Energy in cooperation with the National Institutes of Health (NIH), launched the project. At about the same time, the governments of the United Kingdom, Japan, Russia, France, and Italy initiated their own, similar undertakings, which are coordinated with American efforts.

The purpose of the project is to locate each human gene and determine its specific structure and function. Such knowledge will provide the framework for studies in health, disease, biology, and medicine during the twenty-first century and no doubt will make possible the cures for countless diseases. Although great strides have been made in gene therapy in a relatively short time, its potential usefulness has been limited by lack of scientific data concerning the multitude of functions that genes control in the human body. For gene therapy to advance to its full potential, scientists must discover the biological role of each of these genes and locate each base pair of which they are comprised.

A Progress Report

Scientists participating in the project have identified an average of one new gene a day, and this rate of discovery has increased. At the time of its establishment in 1990 (under the leadership of James D. Watson, who served as director until 1992), HGP was expected to reach completion by 2005. In 2002, however, the project's leadership predicted completion by some time in the following year. Along the way, they had discovered that the human genome, originally believed to include 100,000 to as many as 150,000 genes, actually consists of about 30,000 to 40,000 genes.

Both HGP and a private firm, Celera Genomics (founded 1998), had undertaken the study of the human genome, and in June 2000 the entities jointly reported that they had finished the initial sequencing of the three billion-odd base pairs in the human genome. By that point, researchers also had completed thorough DNA sequences for many other organisms. The basis for the latter undertaking is that humans share many genes with other life-forms. With the completion of initial sequencing, scientists working on the HGP undertook the effort of determining the exact sequence of the base pairs that make them up all human genes. Long before completion, the project had yielded some information. Some of the genes identified through the HGP include one that guides reproduction of the human immunodeficiency virus (HIV), which causes the acquired immunodeficiency syndrome, better known as AIDS (see Infectious Diseases). Researchers also have located a gene that predisposes people to obesity as well as genes associated with such inherited disorders as Huntington disease, Lou Gehrig disease (also called amyotrophic lateral sclerosis, or ALS), and some colon and breast cancers.

A World of Controversy

The HGP has numerous implications—and not all of them, in the minds of some critics, are positive. The NIH inadvertently created cause for concern when, in 1991, it attempted to patent certain forms of DNA. While patenting is touted as a necessary financial incentive for research initiatives, critics maintain that it restricts access to the information generated and to the use of those discoveries. That places a great deal of control in the hands of the private firm that funded the research and may limit the spread of real benefits that result from discovery.

Some scientists and politicians have raised the concern that the ability to produce detailed genetic information on people could give too much power to the people who possess that knowledge. Most states do not have laws protecting citizens against the misuse of genetic information, for instance, by employers and insurers. In the absence of effective legal remedies, genetic testing may be used to bar people from employment or insurance coverage. Insurers may even make mandatory testing a requirement for coverage. Existing laws may not be adequate to protect people's privacy: whereas the individual may be protected from having to provide potentially damaging genetic information, such information still can be obtained by testing the individual's relatives.

Nightmarish Images

The idea of genetic information being used to control a person's destiny calls to mind all sorts of nightmarish images, such as those raised by the movie Gattaca (1997). Set in a dystopian, or anti-utopian, future, the film depicts a character who employs elaborate means to conceal his true identity in order to hold on to a job that otherwise would be forbidden to him because of his DNA. In fact, one does not have to go to fiction to find examples of societies and movements that have used genetics as a form of social control.

The most frightening example of this was Nazi Germany, which practiced mass murder not only of Jews and other ethnic and social groups but also of people who suffered from mental retardation or other "undesirable" traits. The purpose of DNA was discovered only a year before the collapse of the Nazi empire in 1945, and one can only imagine what Adolf Hitler and his minions would have done with this knowledge if they had had access to it. (DNA is one of several scientific and technological concepts that came to fruition at the end of World War II but which Hitler, fortunately, was unable to use to his advantage. Others include rocketry, nuclear weaponry, radar, computers, and television.)

Nazism was actually an especially repugnant version of a movement known as eugenics, which had its origins in the late nineteenth and early twentieth centuries. Based on the idea that populations could be improved by encouraging people with "positive" traits to reproduce while discouraging reproduction among those with less desirable traits, eugenics was at one time a mainstream movement whose adherents included the distinguished U.S. Supreme Court justice Oliver Wendell Holmes, Jr. (1841-1935).

Other Concerns

The specter of eugenics raises the threat that a single human, or a group of humans, could "play God" with the lives of others. Another dramatic fear associated with genetic engineering is the threat that a genetically re-engineered virus could turn out to be extremely virulent, or deadly, and spread. There are other, more mundane questions of ethics: for instance, is it appropriate for scientists to establish private, for-profit corporations to benefit from discoveries they made while working for public-sponsored research institutions? No wonder, then, that the budget for the HGP in the United States includes a small allocation (3% of its total) toward study of the ethical, legal, and social implications (ELSI) of the project. The ELSI Working Group is charged with studying the issues of fairness, privacy, delivery of health care, and education. Meanwhile, there is a vast body of opposition to genetic engineering, biotechnology, and the HGP. And no aspect of the larger subject is more upsetting to certain individuals, as well as special interest groups, as that of cloning.

Cloning

A clone is a cell, group of cells, or organism that contains genetic information identical to that of the parent cell or organism. It is a form of asexual reproduction (see Reproduction), and as such it is not as new as it seems; what is new, however, is humans' ability to manipulate cloning at the genetic level. The first clones produced by humans as long as 2,000 years ago were plants developed from grafts and stem cuttings. By cloning—a process that calls into play complex laboratory techniques and the use of DNA replication—people usually mean a relatively recent scientific advance. Among these techniques is the ability to isolate and copy (that is, to clone) individual genes that direct an organism's development.

The Promise of Cloning

The cloning of specific genes can provide large numbers of copies of that gene for use in genetic and taxonomic research as well as in the practical areas of medicine and farming. In the latter field, the goal is to clone plants with specific traits that make them superior to naturally occurring organisms. For example, in 1985 scientists conducted field tests using clones of plants whose genes had been altered in the laboratory to generate resistance to insects, viruses, and bacteria. New strains of plants resulting from cloning could produce crops that can grow in poor soil or even underwater and fruits and vegetables with improved nutritional qualities and longer shelf lives. A cloning technique known as twinning could induce livestock to give birth to twins or even triplets, and on the environmental front cloning might help save endangered species from extinction.

In the realm of medicine and health, cloning has been used to make vaccines and hormones. It has become possible, by combining two different kinds of cells (such as mouse and human cancer cells), to produce large quantities of specific antibodies, via the immune system, to fight off disease. When injected into the bloodstream, these cloned antibodies seek out and attack disease-causing cells anywhere in the body. By attaching a tracer element to the cloned antibodies, scientists can locate hidden cancers, and by attaching specific cancer-fighting drugs, the treatment dose can be transported directly to the cancer cells.

Experiments in Cloning

The modern era of laboratory cloning began in 1958 when the British plant physiologist F. C. Steward (1904-1993) cloned carrot plants from mature single cells placed in a nutrient culture containing hormones. The first cloning of animal cells took place in 1964, when the British molecular biologist John B. Gurdon (1933-1989) took nuclei from intestinal cells of toad tadpoles and injected them into unfertilized eggs. The cell nuclei in the eggs had been destroyed with ultra-violet light, but when the eggs were incubated, Gurdon found that 1-2% of the eggs developed into fertile, adult toads.

The first successful cloning of mammals occurred nearly 20 years later, when scientists in Switzerland and the United States successfully cloned mice using a method similar to Gurdon's approach. Their method required one extra step, however: after taking the nuclei from the embryos of one type of mouse, they transferred them into the embryos of another type of mouse. The latter served as a surrogate, or replacement, mother. The cloning of cattle livestock was tried first in 1988, when embryos from prize cows were transplanted to unfertilized cow eggs whose own nuclei had been removed. An even greater breakthrough transpired on February 24, 1997, with the birth of a lamb named Dolly in Edinburgh, Scotland. Dolly was no ordinary sheep: she was the first mammal born from the cloning of an adult cell. Thus, she had been produced by asexual reproduction in the form of genetically engineered cloning rather than by anything resembling a normal process. Nonetheless, she proved her own ability to reproduce the old-fashioned way when, on April 23, 1998, she gave birth to a daughter named Bonnie.

Are Humans Next?

Though Dolly's and Bonnie's births excited hopes, they also inspired fears. If large mammals could be cloned, could humans? As early as 1993 an attempt had been made at cloning human embryos as part of studies on in vitro (out of the body) fertilization. The purpose was to develop fertilized eggs in test tubes and then to implant them into the wombs of women having difficulty becoming pregnant. These fertilized eggs, however, did not develop to a stage that was suitable for transplantation into a human uterus. Then, on October 13, 2001, scientists at Advanced Cell Technology in Worcester, Massachusetts, successfully cloned a human embryo. They had not created human life, as it might sound; what they had developed instead was a source for nerve and other tissues that could be harvested for use in medicine and research. Still, the news—overshadowed though it was in America, where people were still reeling from the September 11 terrorist attacks—was earth-shattering. Human cells had been reproduced, and once again it appeared that the production of human clones might be possible.

It is easy to understand how people might respond with alarm to such frightening news with alarm. Such fears have a great deal more to do with Hollywood than they do with science. In fact, the accomplishment of the Massachusetts firm, while impressive from a scientific standpoint, was fairly modest compared with the Frankenstein-like image presented by anti-genetic engineering scaremongers. "Cloned an embryo" actually sounds a great deal more dramatic than what the Massachusetts scientists achieved, with just one embryo reaching the size of six cells before the cells stopped dividing. This is hardly the beginnings of a clone army.

At any rate, the cloning practiced at the Massachusetts firm was therapeutic cloning, involving the production of genetic material for the treatment of specific conditions. It is a far cry from reproductive cloning, which entails implanting a cloned embryo in a uterus—and even that is still a long way from the clichéd image of clones produced in a test tube without any parents other than the biological material used to create them.

Such ideas are related much more closely to those highlighted in Aldous Huxley's 1932 novel Brave New World than they are to scientific realities. And even if humans wanted to develop such technology, it would be many, many years in the future. As for "creating life," to do so is probably not even possible; if it is, such an achievement is about as far off as travel to another solar system. This is not to say that all fears of cloning and genetic engineering are unwarranted; on the contrary, it is good to have a healthy level of skepticism. But it is also good to be an equal-opportunity skeptic and therefore to question ideas in the popular culture—including opposition to genetic engineering.

Where to Learn More

Barash, David P. Revolutionary Biology: The New, Gene-Centered View of Life. New Brunswick, NJ: Transaction Publishers, 2001.

Chadwick, Ruth F. The Concise Encyclopedia of the Ethics of New Technologies. San Diego: Academic Press, 2001.

"Cloning." New Scientist (Web site). <http://www.newscientist.com/hottopics/cloning/>.

Department of Energy Human Genome Program (Web site). <http://www.ornl.gov/hgmis/>.

Genetic Engineering and Cloning: Improving Nature or Uncorking the Genie? (Web site). <http://library.thinkquest.org/19697/>.

Genetics Education Center, University of Kansas Medical Center (Web site). <http://www.kumc.edu/gec/>.

Hyde, Margaret O., and John F. Setaro. Medicine's Brave New World: Bioengineering and the New Genetics. Brookfield, CT: Twenty-First Century Books, 2001.

Judson, Karen. Genetic Engineering: Debating the Benefits and Concerns. Berkeley Heights, NJ: Enslow Publishers, 2001.

National Human Genome Research Institute (Web site). <http://www.nhgri.nih.gov>.

Twenty Facts About the Human Genome. Wellcome Trust (Web site). <http://www.wellcome.ac.uk/en/genome/thgfac.htm>.

Wade, Nicholas. Life Script: How the Human Genome Discoveries Will Transform Medicine and Enhance Your Health. New York: Simon & Schuster, 2001.

The artificial recombination of nucleic acid molecules in the test tube, their insertion into a virus, bacterial plasmid, or other vector system, and the subsequent incorporation of the chimeric molecules into a host organism in which they are capable of continued propagation. The construction of such molecules has also been termed gene manipulation because it usually involves the production of novel genetic combinations by biochemical means. See also Nucleic acid.

Genetic engineering provides the ability to propagate and grow in bulk a line of genetically identical organisms, all containing the same artificially recombinant molecule. Any genetic segment as well as the gene product encoded by it can therefore potentially be amplified. For these reasons the process has also been termed molecular cloning or gene cloning. See also Gene.

Basic techniques

The central techniques of such gene manipulation involve (1) the isolation of a specific deoxyribonucleic acid (DNA) molecule or molecules to be replicated (the passenger DNA); (2) the joining of this DNA with a DNA vector (also known as a vehicle or a replicon) capable of autonomous replication in a living cell after foreign DNA has been inserted into it; and (3) the transfer, via transformation or transfection, of the recombinant molecule into a suitable host.

Isolation of passenger DNA

Passenger DNA may be isolated in a number of ways; the most common of these involves DNA restriction. Restriction endonucleases make possible the cleavage of high-molecular-weight DNA. Although three different classes of these enzymes have been described, only type II restriction endonucleases have been used extensively in the manipulation of DNA. Type II restriction endonucleases are DNAases that recognize specific short nucleotide sequences (usually 4 to 6 base pairs in length), and then cleave both strands of the DNA duplex, generating discrete DNA fragments of defined length and sequence. A number of restriction enzymes make staggered cuts in the two DNA strands, generating single-stranded termini. See also Restriction enzyme.

The various fragments generated when a specific DNA is cut by a restriction enzyme can be easily resolved as bands of distinct molecular weights by agarose gel electrophoresis. Specific sequences of these bands can be identified by a technique known as Southern blotting. In this technique, DNA restriction fragments resolved on a gel are denatured and blotted onto a nitrocellulose filter. The filter is incubated together with a radioactively labeled DNA or RNA probe specific for the gene under study. The labeled probe hybridizes to its complement in the restricted DNA, and the regions of hybridization are detected autoradiographically. Fragments of interest can then be eluted out of these gels and used for cloning. Purification of particular DNA segments prior to cloning reduces the number of recombinants that must later be screened. See also Electrophoresis.

Another method that has been used to generate small DNA fragments is mechanical shearing. Intense sonification of high-molecular-weight DNA with ultrasound, or high-speed stirring in a blender, can both be used to produce DNA fragments of a certain size range. Shearing results in random breakage of DNA, producing termini consisting of short, single-stranded regions. Other sources include DNA complementary to poly(A) RNA, or cDNA, which is synthesized in the test tube, and short oligonucleotides that are synthesized chemically. See also Oligonucleotide.

Joining DNA molecules

Once the proper DNA fragments have been obtained, they must be joined. When cleavage with a restriction endonuclease creates cohesive ends, these can be annealed with a similarly cleaved DNA from another source, including a vector molecule. When such molecules associate, the joint has nicks a few base pairs apart in opposite strands. The enzyme DNA ligase can then repair these nicks to form an intact, duplex recombinant molecule, which can be used for transformation and the subsequent selection of cells containing the recombinant molecule. Cohesive ends can also be created by the addition of synthetic DNA linkers to blunt-ended DNA molecules.

Another method for joining DNA molecules involves the addition of homopolymer extensions to different DNA populations followed by an annealing of complementary homopolymer sequences. For example, short nucleotide sequences of pure adenine can be added to the 3′ ends of one population of DNA molecules and short thymine blocks to the 3′ ends of another population. The two types of molecules can then anneal to form mixed dimeric circles that can be used directly for transformation.

The enzyme T4 DNA ligase carries out the intermolecular joining of DNA substrates at completely base-paired ends; such blunt ends can be produced by cleavage with a restriction enzyme or by mechanical shearing followed by enzyme treatment.

Transformation

The desired DNA sequence, once attached to a DNA vector, must be transferred to a suitable host. Transformation is defined as the introduction of foreign DNA into a recipient cell. Transformation of a cell with DNA from a virus is usually referred to as transfection.

Transformation in any organism involves (1) a method that allows the introduction of DNA into the cell and (2) the stable integration of DNA into a chromosome, or maintenance of the DNA as a self-replicating entity. See also Transformation (bacteria).

Escherichia coli is usually the host of choice for cloning experiments, and transformation of E. coli is an essential step in these experiments. Escherichia coli treated with calcium chloride are able to take up DNA from bacteriophage lambda as well as plasmid DNA. Calcium chloride is thought to effect some structural alterations in the bacterial cell wall. An efficient method for transformation in Bacillus species involves polyethylene glycol-induced DNA uptake in bacterial protoplasts and subsequent regeneration of the bacterial cell wall. Actinomycetes can be similarly transformed. Transformation can also be achieved by first entrapping the DNA with liposomes followed by their fusion with the host cell membrane. Similar transformation methods have been developed for lower eukaryotes such as the yeast Saccharomyces cerevisiae and the filamentous fungus Neurospora crassa. See also Liposomes.

Several methods are available for the transfer of DNA into cells of higher eukaryotes. Specific genes or entire viral genomes can be introduced into cultured mammalian cells in the form of a coprecipitate with calcium phosphate. DNA complexed with calcium phosphate is readily taken up and expressed by mammalian cells. DNA complexed with diethylamino-ethyl-dextran (DEAE-dextran) or DNA trapped in liposomes or erythrocyte ghosts may also be used in mammalian transformation. Alternatively, bacterial protoplasts containing plasmids can be fused to intact animal cells with the aid of chemical agents such as polyethylene glycol (PEG). Finally, DNA can be directly introduced into cells by microinjection. The efficiency of transfer by each of these methods is quite variable.

Introduction of DNA sequences by insertion into the transforming (T)-DNA region of the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens is a method of introducing DNA into plant cells and ensuring its integration. Because of the limitations of the host range of A. tumefaciens, however, alternative transformation systems are being developed for gene transfer in plants. They include the use of liposomes, as well as induction of DNA uptake in plant protoplasts. Foreign DNA has been introduced into plant cells by a technique called electroporation. This technique involves the use of electric pulses to make plant plasma membranes permeable to plasmid DNA molecules. Plasmid DNA taken up in this way has been shown to be stably inherited and expressed.

Cloning vectors

There is a large variety of potential vectors for cloned genes. The vectors differ in different classes of organisms.

Prokaryotes and lower eukaryotes

Three types of vectors have been used in these organisms: plasmids, bacteriophages, and cosmids. Plasmids are extrachromosomal DNA sequences that are stably inherited. Escherichia coli and its plasmids constitute the most versatile type of host-vector system known for DNA cloning. Several natural plasmids, such as ColE1, have been used as cloning vehicles in E. coli. In addition, a variety of derivatives of natural plasmids have been constructed by combining DNA segments and desirable qualities of older cloning vehicles. The most versatile and widely used of these plasmids is pBR322. Transformation in yeast has been demonstrated using a number of plasmids, including vectors derived from the naturally occurring 2μ plasmid of yeast.

Bacteriophage lambda is a virus of E. coli. Several lambda-derived vectors have been developed for cloning in E. coli, and for the isolation of particular genes from eukaryotic genomes. These lambda derivatives have several advantages over plasmids: (1) Thousands of recombinant phage plaques can easily be screened for a particular DNA sequence on a single petri dish by molecular hybridization. (2) Packaging of recombinant DNA in laboratory cultures provides a very efficient means of DNA uptake by the bacteria. (3) Thousands of independently packaged recombinant phages can be easily replicated and stored in a single solution as a “library” of genomic sequences. See also Bacteriophage.

Plasmids have also been constructed that contain the phage cos DNA site, required for packaging into the phage particles, and ColE1 DNA segments, required for plasmid replication. These plasmids have been termed cosmids. The recombinant cosmid DNA is injected into a host and circularizes like phage DNA but replicates as a plasmid. Transformed cells are selected on the basis of a vector drug resistance marker.

Animal cells

In contrast to the wide variety of plasmid and phage vectors available for cloning in prokaryotic cells, relatively few vectors are available for introducing foreign genes into animal cells. The most commonly used are derived from simian virus 40 (SV40). Normal SV40 cannot be used as a vector, since there is a physical limit to the amount of DNA that can be packaged into the virus capsid, and the addition of foreign DNA would generate a DNA molecule too large to be packaged. However, SV40 mutants lacking portions of the genome can be propagated in mixed infections in which a “helper” virus supplies the missing function. See also Adeno-SV40 hybrid virus.

Plant cells

Two systems for the delivery and integration of foreign genes into the plant genome are the Ti plasmid of the soil bacterium Agrobacterium and the DNA plant virion cauliflower mosaic virus. The Ti plasmid is a natural gene transfer vector carried by A. tumefaciens, a pathogenic bacterium that causes crown gall tumor formation in dicotyledonous plants. A T-DNA segment present in the Ti plasmid becomes stably integrated into the plant cell genome during infection. This property of the Ti plasmid has been exploited to show that DNA segments inserted in the T-DNA region can be cotransferred to plant DNA. See also Crown gall.

Applications

Recombinant DNA technology has permitted the isolation and detailed structural analysis of a large number of prokaryotic and eukaryotic genes. This contribution is especially significant in the eukaryotes because of their large genomes. The methods outlined above provide a means of fractionating and isolating individual genes, since each clone contains a single sequence or a few DNA sequences from a very large genome. Isolation of a particular sequence of interest has been facilitated by the ability to generate a large number of clones and to screen them with the appropriate “probe” (radioactively labeled RNA or DNA) molecules.

Genetic engineering techniques provide pure DNAs in amounts sufficient for mapping, sequencing, and direct structural analyses. Furthermore, gene structure-function relationships can be studied by reintroducing the cloned gene into a eukaryotic nucleus and assaying for transcriptional and translational activities. The DNA sequences can be altered by mutagenesis before their reintroduction in order to define precise functional regions.

Genetic engineering methodology has provided means for the large-scale production of polypeptides and proteins. It is now possible to produce a wide variety of foreign proteins in E. coli. These range from enzymes useful in molecular biology to a vast range of polypeptides with potential human therapeutic applications, such as insulin, interferon, growth hormone, immunoglobins, and enzymes involved in the dynamics of blood coagulation. See also Biotechnology.

Finally, experiments showing the successful transfer and expression of foreign DNA in plant cells using the Ti plasmid, as well as the demonstration that whole plants can be regenerated from cells containing mutated regions of T-DNA, indicate that the Ti plasmid system may be an important tool in the genetic engineering of plants. Such a system will help in the identification and characterization of plant genes as well as provide basic knowledge about gene organization and regulation in higher plants. Once genes useful for crop improvement have been identified, cloned, and stably inserted into the plant genome, it will be possible to engineer plants to be resistant to environmental stress, to pests, and to pathogens. See also Breeding (plant); Gene; Gene action; Somatic cell genetics.

Genetic engineering consists of a set of methods to change the genes of an organism so that proteins produced by that organism differ in type or quantity from those produced by a "wild" organism -- one that has not been altered. Although people have been causing such changes in organisms since domestication began about 10,000 years ago, the phrase genetic engineering conventionally refers to a set of techniques that became possible only in 1973.

It is easy to think that genetic engineering was the inevitable outcome of the Watson-Crick explanation of the mechanism of heredity in 1953, but that is not really the case. While knowledge of DNA structure and the genetic code is essential to genetic engineering, the path that led to practical results was separate from the path that led to understanding. Along the way, of course, both paths interacted constantly. The path that led to practical applications could not have been predicted in advance, as almost all of the important discoveries were previously unsuspected by any scientist.

The start of the path toward genetic engineering occurred in 1952 when Joshua Lederberg discovered that bacteria, like some protists, conjugate to exchange genetic material. This behavior, much like sex in multicellular organisms, led Lederberg to perceive that there are two populations of bacteria, which he called M and F. The F population contains a body that he called a plasmid. After conjugation, the F bacterium passes the plasmid on to the M bacterium, with which it has conjugated. (It would appear that Lederberg had his sexes backward, but that is not essential to what follows.) This discovery was completely surprising.

The next year William Hayes established that the plasmid consists of genetic material. By then it was clear that genes are DNA; therefore, plasmids are rings of DNA floating free of the main DNA of a bacterium.

About the same time, an apparently unrelated situation became a major problem. Both the sulfa drugs and the antibiotics of the late 1930s and 1940s were, in the 1950s, beginning not to work as well as they had. Many bacteria were becoming resistant to those drugs. Epidemics, especially in hospitals, could no longer be controlled. Many scientists studied the problem. In 1959 Japanese scientists discovered that the genes for drug resistance were carried on plasmids, and therefore passed from bacterium to bacterium. Within a given bacterium, the plasmids multiplied, so there were plenty of copies to pass around. Inserting a few drug-resistant bacteria into a colony of bacteria that showed no resistance resulted in short order in a colony that was completely resistant.

It is not surprising that some bacteria had plasmids that protected against these drugs. Antibiotics are natural substances found in the environment, so some bacteria have evolved defenses against them. The increase in the amount of antibiotics caused by human intervention led to the resistant bacteria passing the plasmids around to larger populations.

In the meantime, another line of research was also leading toward genetic engineering. Starting right after World War II, a number of biologists made an intensive study of viruses that infect bacteria, which are collectively called bacteriophages, or just phages. This line of research demonstrated that genes are part of DNA and not part of a protein, as had previously been suspected. As early as 1946 Max Delbrück and Alfred Hershey independently showed that the genes from different phages could spontaneously combine. Werner Arber studied the mutation process in phages in detail. In the process he discovered that bacteria resist phages by splitting the phage DNA with enzymes. Subsequent recombination of split genes was a consequence of this. By 1968 Arber had located the enzymes that split DNA at specific locations. The split ends are "sticky"; that is, different genes that have been split at the same location by one of these restriction enzymes, as they came to be called, will recombine when placed together in the absence of the enzyme. The resulting product is called recombinant DNA.

The following year, 1969, Jonathan Beckwith and coworkers became the first to isolate a single gene. It was a bacterial gene for a part of the metabolism of sugar.

In 1973 Stanley Cohen and Herbert Boyer combined the restriction enzymes with plasmids with isolation of specific genes to introduce genetic engineering. They cut a chunk out of a plasmid found in the bacterium Escherichia coil and inserted into the opening a gene from a different bacterium. Then they put the plasmid back into the bacteria E. coli, where copies were made and transferred to other bacteria. Within months other scientists repeated the trick, inserting genes from fruit flies and frogs into E. coli.

Not all scientists thought this was a good thing. In July 1974, Paul Berg and other biologists met under the auspices of the U.S. National Academy of Sciences to draw up guidelines that would prohibit certain kinds of genetic engineering.

Since 1974, the tension between those who are rapidly advancing genetic engineering and those who worry about where it is going has continued. By the 1980s, the genetic engineers were producing useful products from bacteria and yeasts, including human growth hormone, human insulin, and a vaccine for hepatitis B. All these are made in tanks in controlled environments and have ceased to evoke much resistance from scientists or the public. There has been more resistance to experiments in which genetically engineered bacteria are released in the environment, although a number of small-scale releases have so far not resulted in known environmental damage. Another source of concern relates to the safety of the many farm crops that have been modified by genetic engineering.

In one area, the progress of genetic engineering has been frustratingly slow. From the beginning of the new technique, there has been hope that it could be used to cure human genetic diseases. So far, that has not proved practical in most cases, although there has been some limited success.

Genetic Engineering is the deliberate manipulation of an organism's genetic makeup to achieve a planned and desired result. Proponents of genetic engineering consider it an extension of the selective breeding practiced for thousands of years in the domestication of agricultural products and animals. The genesis of modern biotechnology, most scholars agree, came in the early 1970s with the advent of recombinant DNA (rDNA). Since biotechnology often refers to the use of organisms in agriculture, industry, or medicine, its origins can be traced back to the use of yeast for baking bread and the fermentation of alcohol. The impact of contemporary genetic engineering and biotechnology affects nearly every area of human activity. The introduction of rDNA engineering has revolutionized our relationship to the organic world and to ourselves, demanding a reconsideration of our values, our notion of progress, and the morality of scientific research.

The History of Genetic Engineering

Genetic engineering owes its existence to the developments in molecular genetics, virology, and cytology that culminated in the determination of the structure of DNA by James Watson and Francis Crick in 1953. Building on research involving bacteriophages (a bacterial virus), Joshua Lederberg, a geneticist at the University of Wisconsin, found that bacteria can transfer genetic information through plasmids, small mobile pieces of DNA that exist independent of the chromosomes. In the 1950s, Lederberg pioneered the earliest techniques in genetic engineering, shuffling genetic material between bacterial cells. After the identification of restriction enzymes capable of "cutting" DNA in specific locations in 1968, scientists were able to insert foreign DNA directly into bacterial cells. The discovery that the foreign DNA would naturally bond with the host DNA, made it possible to splice together genes from multiple organisms, the technique used in recombinant DNA engineering. Although highly complicated, rDNA engineering can be simply explained: genetic material from the donor source is isolated and "cut" using a restriction enzyme and then recombined or "pasted" into the genetic material of the receiver. By 1971, advanced transplantation techniques had been developed and rDNA techniques using the restriction enzyme EcoRi were operable the following year, leading to the first experiments in genetic engineering.

In 1973, Stanford biochemist Stanley Cohen under-took one of the first rDNA experiments, inserting a piece of bacterial DNA into Escherichia coli (E. coli), a bacterium found in the human intestine. However, the research soon became controversial, particularly when American molecular biologist Paul Berg designed an experiment to insert DNA from simian virus #40 (sv40)—a known cancer-causing agent—into E. coli. As word of the daring procedure spread, the public was captivated and fearful, afraid that a genetically engineered virus, inured to antibiotics and carried in a common bacterium, could escape and cause an epidemic. Hoping to diffuse fears of a potential biohazard and maintain control of their research, over one hundred and fifty molecular biologists and related specialists met at the Asilomar Conference Center in Monterey, California, in late February 1975. The conference represented an extraordinary moment in the history of science, as the research community, recognizing its social responsibility, officially adopted a moratorium until appropriately safe procedures and guidelines could be developed. The conference ultimately resulted in the "National Institutes of Health Guidelines for Research Involving rDNA Molecules" and an ongoing National Institute of Health rDNA Advisory Committee (RAC)founded in 1974.

Yet the guidelines only increased public concern over genetic engineering. Critics charged that attempts to splice genes together from different organisms were akin to "playing God" and could result in dangerous and immoral hybrids. Adopting the literary example of "Dr. Frankenstein's monster" as an appropriate symbol of misguided science, opponents of rDNA engineering converged on research laboratories and public meetings. An attempt to build a recombinant laboratory at Harvard University set off such a firestorm that local politicians created a review board to assess potential risks, eventually requiring more stringent controls than those set by the NIH. By 1977, protests of rDNA facilities had spread to other campuses—the University of California San Diego, the University of Wisconsin, the University of Michigan, and the University of Indiana—while the state legislatures of New York, New Jersey, and California held public hearings. However, it was the resolution of an old court case and the introduction of a new form of rDNA engineering that ultimately created the greatest controversy.

In a monumental decision handed down on 16 June 1980, the United States Supreme Court held in Diamond v. Chakrabarty that man-made life forms were subject to patent laws and protection. The decision resolved a longstanding issue on patents and organic material, as the case dated to 1972, when Ananda Chakrabarty, a researcher at General Electric, applied for a patent on a form of Pseudomonas bacteria bred (but not genetically engineered)to digest oil slicks. By a narrow five to four margin the court construed the Patent Act, originally drafted by Thomas Jefferson, so as to include all products of human invention, relying on a 1952 Senate report that recognized as patentable "anything under the sun that is made by man." More than any other single event, the ruling galvanized many mainstream religious communities and environmental groups, eventually resulting in a letter of protest to President Carter and an indepth review by the President's Commission for the Study of Ethical Problems in Medicine and Biomedical and Behavioral Research (1980–1983). The commission's report, issued in 1982 and entitled Splicing Life: The Social and Ethical Issues of Genetic Engineering with Human Beings, emphasized the importance of rDNA engineering to biomedical progress and American industries, arguing that it was best that the research be conducted under the auspices of government regulation and control. However, while the study resolved anxiety over rDNA engineering and patenting, proponents of genetic engineering still had to address concerns over the development of "germ-line" engineering, a controversial procedure that allowed scientists to literally create new strains of organisms.

Germ-line engineering differs from rDNA engineering in that the donor genes are inserted into a "germ," or reproductive cell, thereby permanently altering the genetic makeup of the organism's descendants. For example, in 1982, Ralph Brinster of the University of Pennsylvania Veterinary School inserted the gene that produces rat growth hormone into mouse embryos. The resulting strain of mice, dubbed "super mice" by the press, expressed the gene and thus grew into a substantially larger and more powerful new breed of mouse. Critics of germ-line engineering quickly denounced the technique as immoral and argued it was a form of "anthropomorphic Lamarckism." Jean-Baptiste de Lamarck, a nineteenth-century French naturalist, had proposed that traits acquired during an organism's lifetime were passed on to its progeny—an idea refuted by Darwinian evolutionary theory. Yet, in germ-line engineering, traits acquired during the organism's lifetime are passed on, but only those traits deemed necessary or desirous by man. Environmental groups also denounced germ-line engineering because of "biosafety" concerns, fearing that genetically engineered species, which would possess a distinct advantage over nonengineered species, could upset the globe's finely tuned ecological systems. However, because most politicians, scientists, and manufacturers believed the potential benefits from rDNA and germ-line engineering outweighed its potential dangers, the protests were overshadowed by the development of a biotechnology industry based on genetic engineering.

Contemporary Applications of Genetic Engineering

The decision to allow patents on genetically engineered organisms, combined with the commission's sanction of rDNA engineering, and a national commitment to biomedical progress, led to tremendous growth in the biotechnology industry. In 1975, only five biotech companies participated in the Asilomar conference, by 1980 the number of similar companies had increased to one hundred. Today there are over 1,300 companies involved in genetic engineering, many of which are located in the United States, a clear indication of the rapid growth of the American biotechnology sector and the applicability of the powerful new techniques. Indeed, genetic engineering influences nearly every area of human activity, including agriculture and aquaculture, industry and environmental remediation, and the development of medicines and therapies.

Although agriculture has been one of the most successful industries in utilizing genetic engineering, the techniques have also made an impact in other areas of food production. In 1990, Chymosin, an enzyme necessary for cheese production, became the first genetically engineered food product to go to market. A few years later, in 1994, the Monsanto Company created a bovine growth hormone designed to stimulate milk production, a hormone now estimated to be given to 30 percent of dairy cows. The same year, the "Flavr-Savr" tomato developed by Calgene passed the Food and Drug Administration standards for genetically engineered foods and also went to market. Like many transgenic foods, the "Flavr-Savr" was designed to have increased shelf life and resist spoilage, although disputes regarding labeling and advertisements combined with high production costs caused the company to discontinue the product in the late 1990s. Nonetheless, genetic engineering is integrated into agriculture production; researchers estimate that as of 2001, nearly one-third of the corn and one half of the soybeans grown in the United States were transgenic. A study conducted in 2000 by the Grocery Manufacturers of America reported that the majority of processed foods sold in America contained transgenic ingredients. To help develop aquaculture, researchers at Johns Hopkins University have taken a gene from flounder and inserted it into both trout and bass in the hopes of making the fish more resistant to cold climates, thus increasing commercial and sport fishing.

Genetic engineering also has substantial applications in many other industries from plastics and energy to the new field of bioremediation. In 1993, Chris Sommerville, director of plant biology at the Carnegie Institute in Washington, D. C., successfully inserted plastic-making genes into a plant; the Monsanto Company hopes to market a cotton/polyester plant early in the twenty-first century. Scientists at numerous biotech companies are currently working on strains of E. coli bacteria capable of transforming agricultural refuse into ethanol, an efficient and clean source of energy. Genetic engineering is also aiding environmental clean-up through the emerging field of bioremediation—the use of organisms to reduce waste. Bacteria were employed to help with the Exxon Valdez oil spill in 1989, while scientists at the Institute for Genomic Research are among those hoping to engineer microbes that can detoxify waste, including radioactive materials. However, the fastest growing, and one of the most controversial, fields of biotechnology is applied human genetics, which includes transgenic medicines, xenotransplantation, and human gene therapy.

In 1982, Eli Lilly and Company began marketing bacterial-produced insulin, the first transgenic commercial product and an excellent marker of the industry's progress. Today, the vast majority of insulin used by Americans diabetics is genetically engineered and over 300 transgenic proteins and medicines are currently in production, many of which are made by animals. Indeed, animal "pharming" has been central to biomedical research and development since the introduction of genetic engineering; in 1988, Harvard University patented the "oncomouse," strains of mice missing or carrying specific genes and used in cancer research. In 1996, Genzyme Transgenics created a goat capable of producing anti-thrombin, an experimental anticancer drug; the following year PPL Therapeutics engineered a calf whose milk contains proteins necessary for nursing babies, including those born prematurely. Human hemoglobin, a protein essential for oxygen transportation in the bloodstream, can now be harvested from genetically engineered pigs. Transgenic pigs are also used in xenotransplantation, the transference of organs or parts from nonhuman species to humans. Nextran, a leading biotech company, hopes to use genetically engineered pig livers as temporary external reservoirs for patients suffering from acute liver failure. In the future, researchers hope that these transgenic medicines and proteins will help supplement human gene therapy, one of the boldest and most ethically and medically problematic areas of genetic engineering.

The history of human gene therapy is one of great promise and success mixed with controversy and stringent regulation. In the early 1980s, Martin Cline, a medical researcher at the University of California in Los Angeles, performed rDNA procedures in Italy and Israel on patients afflicted with hereditary blood disorders. Cline's unauthorized experimentation, although legal because the countries lacked genetic regulations, ultimately cost him funding and a department chairmanship. In response, the RAC established the Human Gene Therapy Subcommittee in 1984 to issue protocols and review applications. Years later, in 1990, researchers at the National Institute of Health (NIH)attempted the first approved human gene therapy for Ashanti DeSilva, a young girl forced to live inside a "bubble" because of severe combined immune deficiency, or ADA. As in most cases of human gene therapy, the researchers removed cells from the patient, genetically engineered the desired changes, and then replaced the cells. However, for ADA, as for most diseases, gene therapy offers only treatment, not a cure, as the procedure must be repeated periodically. Nonetheless, the success of Ashanti's procedure stimulated human gene therapy research; in 1992, Bernadine Healy, then director of the NIH, approved a "compassionate use exemption" to increase access to promising gene therapy trials for critically ill patients. Within a year, procedures had been approved for familial hypercholesterolemia, cystic fibrosis, and Gaucher's disease, and trials for cancer, AIDS, Parkinson's, Alzheimers, arthritis, and heart disease were being conducted. Unfortunately, the 1999 death from liver disease of Jesse Gelsinger, an eighteen-year-old student taking part in a University of Pennsylvania gene therapy trial, led to questions regarding the safety of established protocols, as the fatality resulted from a common immune reaction to the adenovirus vector (see Genetics) that the researchers could have easily anticipated.

Although genetic engineering remains in its infancy, the rapid development of the science and its related techniques has generated considerable disagreement in the attempt to address its moral and legal implications. The birth of the sheep "Dolly" in 1997, the first cloned adult mammal, led to debates over the sanctity of life and the individual, while the advent of human gene therapy has revived fears of eugenics programs and genetically engineered "designer" children. The marketing of transgenic foods stimulated the growth of an "organic" agricultural industry and created ongoing international disputes over patent rights, truth-in-labeling claims, and restrictions on genetically engineered imports. Some critics fear that xenotransplantation will promote the transference of animal diseases to humans, while others decry the use of animals simply for the benefit of mankind. The development of stem cell research, promising because the embryonic cells can be manipulated to become nearly any type of cell in the body, has led to protests by many pro-life organizations over the use of embryonic or fetal tissue; in August 2001, President Bush declared that only a limited number of cell lines were acceptable for federal research funding. Whether involved in human gene therapy, xenotransplantation, industry, or agriculture, genetic engineering and biotechnology will no doubt continue providing astounding advancements alongside heated controversy and debate well into the future.

Bibliography

Bud, Robert. The Uses of Life: A History of Biotechnology. New York: Cambridge University Press, 1993.

Fiechter, A., ed. History of Modern Biotechnology. 2 vols. Berlin: Springer Verlag, 2000.

Rifkin, Jeremy. The Biotech Century: Harnessing the Gene and Remaking the World. New York: Putnam, 1998.

Shannon, Thomas A., ed. Genetic Engineering: A Documentary History. Westport, Conn. : Greenwood Press, 1999.

Von Wartburg, Walter P., and Julian Liew. Gene Technology and Social Acceptance. Lanham, Md. : University Press of America, 1999.

—J. G. Whitesides

Genetic engineering, also known popularly as molecular cloning or gene cloning, is the artificial recombination of nucleic acid molecules in the test tube, their insertion into a virus, bacterial plasmid, or other vector system, and the subsequent incorporation of the chimeric molecules into a host organism in which they are capable of continued propagation. The construction of such molecules has also been termed gene manipulation because it usually involved the production of novel genetic combinations by biochemical means.

Genetic engineering techniques include cell fusion and the use of recombinant DNA (RNA) or gene-splicing. In cell fusion, the tough outer membranes of sperm and egg cells are stripped off by enzymes, and then the fragile cells are mixed and combined with the aid of chemicals or viruses. The result may be the creation of a new life form from two species. Recombinant DNA techniques transfer a specific genetic activity from one organism to the next through the use of bacterial plasmids (small circular pieces of DNA lying outside the main bacterial chromosome) and enzymes, such as restriction endonucleases (which cut the DNA strands); reverse transcriptase (which makes a DNA strand from an RNA strand); DNA ligase (which joins DNA strands together); and tag polymerase (which can make a double-stranded DNA molecule from a single stranded "primer" molecule). The process begins with the isolation of suitable DNA strands and fragmenting them. After these fragments are combined with vectors, they are carried into bacterial cells where the DNA fragments are "spliced" on to plasmid DNA that has been opened up. These hybrid plasmids are now mixed with host cells to form transformed cells. Since only some of the transformed cells will exhibit the desired characteristic or gene activity, the transformed cells are separated and grown individually in cultures. This methodology has been successful in producing large quantities of hormones (such as insulin) for the biotechnology industry. However, it is more difficult to transform animal and plant cells. Yet the technique exists to make plants resistant to diseases and to make animals grow larger. Because genetic engineering interferes with the processes of heredity and can alter the genetic structure of our own species, there is much concern over the ethical ramifications of such power, as well as the possible health and ecological consequences of the creation of these bacterial forms. Some applications of genetic engineering in the various fields are listed below:

Agriculture: Crops having larger yields, disease- and drought-resistancy; bacterial sprays to prevent crop damage from freezing temperatures; and livestock improvement through changes in animal traits.

Industry: Use of bacteria to convert old newspaper and wood chips into sugar; oil- and toxin-absorbing bacteria for oil spill or toxic waste clean-ups; and yeasts to accelerate wine fermentation.

Medicine: Alteration of human genes to eliminate disease (experimental stage); faster and more economical production of vital human substances to alleviate deficiency and disease symptoms (but not to cure them) such as insulin, interferon (cancer therapy), vitamins, human growth hormone ADA, antibodies, vaccines, and antibiotics.

Research: Modification of gene structure in medical research, especially cancer research. Food processing: Rennin (enzyme) in cheese aging.

Genetic engineering involves the directed alteration of an organism's DNA (deoxyribonucleic acid)—that is, its genetic material. This technology has been applied to microbes, plants, and animals, and consequently used to modify foods, animal feedstuffs, and food-processing reagents.

Domestication and improvement of plants and animals for agriculture initially relied on identification of individuals with desirable characteristics from among natural populations. Applying knowledge of genetics to the breeding of plants and animals resulted in more rapid progress and remains vitally important to agricultural development. Traditional breeding, however, is constrained by the boundaries of sexual compatibility, which limits the choice of parents that can be used as sources of genes and traits to improve a specific crop or animal to those that can produce progeny through sexual reproduction. Genetic engineering expands the source of genes that can be used to modify the characteristics of plants and animals.

Technology of Genetic Engineering

Genetic engineering requires three fundamental technologies: the ability to isolate and modify the DNA of specific individual genes; an understanding of the mechanisms that regulate how genes function and how these can be manipulated; and the capacity to transfer genes into an organism. These have all been developed following the discovery of the structure of DNA in 1953. Genetic engineering of microbes was first reported in 1973, followed in the next decade by similar achievements in plants and animals. Because DNA is the genetic material in all organisms, genes for genetic engineering can be taken from any source, or even synthesized. Modification of genes may be necessary, particularly in regions that control how they operate, in order for the genes to function effectively in the recipient organism. Agrobacterium tumefaciens, a bacterium that transfers DNA into plant cells as part of its normal life cycle, is used commonly to transfer genes into plants, although other methods such as the "gene gun" also have been developed. Genetically engineered plants are technically "transgenic organisms," as they contain transferred genes. However, they are frequently referred to as "genetically modified organisms," or GMOs, and the products derived from them are described as "genetically modified," or GM foods. These terms can be confusing, as essentially all cultivated plants have been genetically modified through breeding and selection—for example, the many varieties of cultivated onions possess numerous qualities that distinguish them from each other and especially from the wild onions from which they originated.

Application of Genetic Engineering in Agriculture

The first genetically engineered crops were planted on a large scale in 1996. By 2001 more than fifty million hectares were planted worldwide with transgenic crops. The first generation of these crops has been altered in ways that improve the efficiency of crop production by modifying the tolerance of plants to herbicides and insect pests. Broad-spectrum herbicides are able to kill almost all plants. A prerequisite for using chemicals to control weeds in a crop is that the crop itself must be resistant to the herbicide. Genetic engineering has been used to develop plants (specifically soybean, canola, corn, and cotton) with resistance to two broad-spectrum herbicides, glyphosate and glufosinate, which are sold under the trademarks Roundup and Liberty, respectively. Glyphosate-tolerant soybeans have been adopted rapidly in some countries, notably the United States and Argentina, and accounted for approximately 46 percent of the soybean acreage worldwide in 2001. Herbicide use has not declined in these crops but the specific herbicides that are used have changed.

Insect pests can damage crops during the growing season and also after harvest. A variety of methods, including cultural practices and insecticides, are used to control insect damage. Genetic engineering has provided novel approaches to this problem. The bacterium Bacillus thuringiensis (Bt) produces proteins that are toxic to some types of insects, and Bt spores have been used as insecticides for decades. Genes encoding Bt toxin proteins have been isolated, modified so they function in plants, and transferred into crop plants including corn, potato, and cotton. These engineered Bt crops are more resistant to such insects as the European corn borer, Colorado potato beetle, and cotton bollworm than are their nonengineered counterparts. The introduction of Bt cotton has resulted in reduced use of insecticides on this crop in some regions of the United States. Growers of Bt crops are required to plant a portion of their acreage with varieties that do not carry the Bt gene, in an effort to delay the development of insect populations with resistance to Bt toxins.

The Flavr Savr tomato, developed in the 1980s by Calgene, a biotechnology company in California, was the first food produced from a genetically engineered plant. These tomatoes ripened more slowly and had an extended shelf life. However, for a number of reasons—including production problems and consumer skepticism—this product was not a commercial success and was withdrawn in 1996, after less than three years on the market. Melons and raspberries have also been engineered to have delayed ripening but have not been produced commercially. Transgenic papayas with resistance to ring spot virus also have been developed. These were grown successfully in Hawaii, where the papaya industry was devastated by this debilitating disease. A similar approach was used to produce virus-resistant summer squash and against other viruses affecting a wide variety of foodstuffs.

The first generation of transgenic crops for the most part were designed to improve the efficiency of crop production, an ongoing objective for genetic engineers. Additionally, the techniques of genetic engineering can be used to alter the nutritional composition of foods. The transfer into rice of three genes that function to produce beta-carotene in the seed resulted in "golden rice." Once consumed, beta-carotene can be converted to vitamin A, the degree of this conversion being dependent upon a number of factors that relate to the source of the beta-carotene, the diet, and the individual consumer. In less-developed countries, vitamin A deficiency is widespread among those with a restricted diet, and is responsible for increased mortality and blindness in children. Although the efficacy of transgenic rice in reducing disease has not been established, it demonstrates the potential use of genetic engineering for nutritional enhancement in many crops. Other applications of genetic engineering of animal and human foods include removing allergens from foods such as peanuts, increasing the level of essential vitamins and nutrients in foods, and producing foods possessed of vaccines and other beneficial compounds.

Genetically engineered microbes also are used to produce proteins for food processing. Chymosin (or rennin), an enzyme used in cheese production, traditionally is obtained from the stomach of veal calves. However, the gene encoding this enzyme was transferred into microbes, and the enzyme now can be produced in bulk by purifying it from large microbe cultures. Chymosin prepared from transgenic microbes has more predictable properties than the animal product and is used to produce more than fifty percent of hard cheeses in the United States. Other enzymes used in food processing are produced by similar methods. For example, bovine growth hormone (BGH) is produced in large quantities from transgenic microbes and is given to cows to increase milk production.

Regulation of Genetic Engineering

In the United States, three federal agencies—Food and Drug Administration (FDA), Environmental Protection Agency (EPA), and Department of Agriculture (USDA) —are involved in regulating transgenic crops. Similar systems are in place in other countries as well. Companies that have developed this technology generally are supportive of the current regulatory framework. Nevertheless, the development of transgenic crops and the introduction of foods that contain products from these plants in the 1990s generated tremendous controversy, notably in Europe. Proponents of genetic engineering have argued that the addition of one or two well-characterized genes into crop plants that have a history of safe use is unlikely to affect materially the properties of these plants. Opponents suggest that this technology has not been tested adequately and the public should not be exposed to unknown and unnecessary food-based risks.

Safety concerns include the possibility that this technology will reduce the nutritional content of foods and introduce novel allergens or other toxins into foods. Opponents have sought more extensive testing and mandatory labeling of products that contain genetically engineered foods so that consumers can choose whether or not to eat such items. The impact of transgenic crops on the environment also has been questioned. Pests are likely to develop resistance to toxins produced by transgenic plants, raising doubts about the sustainability of this approach. However, transgenic technology also has the potential to reduce the use of chemical pesticides for crop production, which most regard as a positive development. Transfer of genes from engineered crops to other plants might also occur—for example, making weeds resistant to a specific herbicide or expanding the range of a plant so that it can grow in new locations.

This new technology also brings forth social, economic, and ethical issues, many of which are reflected by a wide political debate. One subject of concern is that most of the technology enabling genetic engineering of crop plants is controlled by a small number of companies. Much of this control is achieved through ownership of intellectual property, such as patents on genes, methods to produce transgenic plants, and the plant material that is the basis for crop improvement. Companies that manage agricultural inputs, such as seeds, pesticides, and fertilizers, as well as food processing and retail operations, function increasingly on a global scale. Opponents of globalization have criticized genetic engineering as one factor that is contributing to this trend and have expressed concern that both farmers and consumers will have limited choice in who supplies their needs. Opposition to genetic engineering also has come from religious groups who believe that tampering with genes in this way is unnatural—that is, inconsistent with the divine domain of nature—and should not be allowed.

Development of methods to genetically modify plants that extend beyond the limits of normal sexual reproduction has the potential to change many aspects of food production. Some of the first generations of products of this technology were adopted readily by most farmers but, as with other new technologies, there are many opponents. If this technology eventually receives widespread acceptance, it is likely that genetically engineered products will be found in almost everything that humans and domesticated animals eat.

Bibliography

Charles, Daniel. Lords of the Harvest: Biotech, Big Money, and the Future of Food. Cambridge, Mass.: Perseus Publishing, 2001. A history of the development of agricultural biotechnology and genetically engineered foods.

Colorado State University. Transgenic Crops: An Introduction and Resource Guide. Available at http://www.colostate.edu/programs/lifesciences/TransgenicCrops/

Ervin, David, Sandra Batie, Rick Welsh, Chantal Carpentier, Jacqueline Fern, Nessa Richman, and Mary Schulz. Transgenic Crops: An Environmental Assessment. Morrilton, Ark.: Winrock International, 2000. Available at http://www.winrock.org/Transgenic.pdf

Nuffield Council on Bioethics. Genetically Modified Crops: The Ethical and Social Issues. London: Nuffield Council on Bioethics, 1999. A report from the United Kingdom that addresses consumer issues.

Pew Initiative on Food and Biotechnology. Harvest on the Horizon: Future Uses of Agricultural Biotechnology. Washington D.C.: Pew Initiative, 2001. Available at http://pewagbiotech.org/research/harvest/

Watson, James, Michael Gilman, Jan Witkowski, and Mark Zoller. Recombinant DNA. 2nd ed. New York: W. H. Freeman, 1992. A detailed description of the science behind genetic engineering.

—Peter Goldsbrough

The human manipulation of the genetic material of a cell.

Genetic engineering involves isolating individual DNA fragments, coupling them with other genetic material, and causing the genes to replicate themselves. Introducing this created complex to a host cell causes it to multiply and produce clones that can later be harvested and used for a variety of purposes. Current applications of the technology include medical investigations of gene structure for the control of genetic disease, particularly through antenatal diagnosis. The synthesis of hormones and other proteins (e.g., growth hormone and insulin), which are otherwise obtainable only in their natural state, is also of interest to scientists. Applications for genetic engineering include disease control, hormone and protein synthesis, and animal research.

International Codes and Ethical Issues for Society

An international code of ethics for genetic research was first established in the World Medical Association's Declaration of Helsinki in 1964. The guide prohibited outright most forms of genetic engineering and was accepted by numerous U.S. professional medical societies, including the American Medical Association (AMA).

In 1969 the AMA promulgated its own ethical guidelines for clinical investigation, key provisions of which conflicted with the Helsinki Declaration. For example, the AMA guidelines proposed that when mentally competent adults were found to be unsuitable subjects for genetic engineering studies, minors or mentally incompetent subjects could be used instead. The Helsinki Declaration did not condone testing on humans.

The growth of genetic engineering in the 1970s aroused international concern, but only limited measures were taken by governments and medical societies to control it. Concern focused on the production of dangerous bacterial mutants that could be used for harmful eugenics tools or weapons. The Genetic Manipulation Advisory Group was established in England based on the recommendations of a prominent medical group, the Williams Committee. Scientists were required to consult this group before carrying out any activity involving genetic manipulation in England. Additional measures required scientific laboratories throughout the world to include physical containment labs to prevent manipulated genes from escaping and surviving in natural conditions. These policies were subsequently adopted in the United States.

The Breakdown of Regulation: Genetic Inventions and Patents in the United States

In 1980 the Supreme Court created an economic incentive for companies to develop genetically engineered products by holding that such products could be patented. In Diamond v. Chakrabarty, 447 U.S. 303, 100 S. Ct. 2204, 65 L. Ed. 2d 144, the Court held that a patent could be issued for a novel strain of bacteria that could be used in the cleanup of oil spills. In 1986, the U.S. Department of Agriculture approved the sale of the first living genetically altered organism. The virus was used as a pseudorabies vaccine, from which a single gene had been cut. Within the next year, the U.S. Patent and Trademark Office announced that nonnaturally occurring, nonhuman, multicellular living organisms, including animals, were patentable under the Patent Act of 1952 (35 U.S.C.A. § 101).

The Department of Agriculture formally became involved in genetic engineering in April of 1988, when the Patent and Trademark Office issued the first animal patent, granted on a genetically engineered mouse used in cancer research. U.S. scientists began experiments with the genetic engineering of farm animals, such as creating cows that would give more milk, chickens that would lay more eggs, and pigs that would produce leaner meat. These developments only raised more objections from critics who believed that genetic experimentation on animals violated religious, moral, and ethical principles. In spite of the controversy, the U.S. House of Representatives approved the Transgenic Animal Patent Reform bill on September 13, 1988. The bill would have allowed exempted farmers to reproduce, use, or sell patented animals, although it prohibited them from selling germ cells, semen, or embryos derived from animals. However, the Senate did not vote on the act and so it did not become law.

Significant State Laws

Certain states have passed laws restricting genetic engineering. By the early 1990s, six states had enacted laws designed to curb or prohibit the spread of genetically engineered products in the marketplace (see Ill. Ann. Stat. ch. 430, § 95/1 [Smith-Hurd 1995]; Me. Rev. Stat. Ann. tit. 7, § 231 et seq. [West 1995]; Minn. Stat. Ann. § 116C.91 et seq. [West 1995]; N.C. Gen. Stat. § 106-765-780 [Supp. 1991]; Okla. Stat. Ann. tit. 2, §§ 2011-2018 [West 1996]; Wis. Stat. Ann. § 146.60 [West 1996]). North Carolina's law sets the most comprehensive restrictions on genetic engineering. Resembling the earlier measures proposed by organizations such as England's Genetic Manipulation Advisory Group, it requires scientists to hold a permit for any release of a genetically engineered product outside of a closed-containment enclosure. The North Carolina statute has been cited as a possible model for advocates of comprehensive federal regulations.

Recent Developments

In the mid 1990s the international guidelines established by the Declaration of Helsinki were modified to allow certain forms of cell manipulation in order to develop germ cells for therapeutic purposes. Scientists are also exploring genetic engineering as a means of combating the HIV virus.

In 1997 the cloning of an adult sheep by Scottish scientist, Ian Wilmut, brought new urgency to the cloning issue. This breakthrough (prior cloning had been successful only with immature cells, not those from an adult animal) raised the prospect of human cloning and prompted what is likely to be a continuing ethical and legal debate on how to regulate cloning.

See: genetic screening.

• 1951 J. Williamson Dragon's Island № 180: I was expecting to find that mutation lab filled with some sort of apparatus for genetic engineering.
• 1972 New York Times (May 1) № 39/1: The ethical implications of this and other experiments in "genetic engineering" — including attempts to produce genetically identical copies of individuals — should be thoroughly explored before the work is applied to man.
• 1993 Super Marketing (Feb.) № 23/2: Four American companies are going ahead with genetic engineering in tomatoes aimed at providing longer shelf-life.
• 2001 M. Pollan Botany of Desire (2001) № 188: With genetic engineering, human control of nature is taking a giant step forward.

The manipulation of DNA to produce new types of organisms, usually by inserting or deleting genes.

• Genetics, Heredity, and Evolution - genetic engineering: incorporation of new genes into an organism to alter it
• Business and Science of Agriculture - genetic engineering: technique of producing new, improved hybrid species by cutting and splicing of genetic units of DNA from different organisms
• Cultural Movements, Events, and Institutions - genetic engineering: biological creation of improved species through recombining of spliced DNA from different species

For a non-technical introduction to the topic, see Introduction to genetics. For the song by Orchestral Manoeuvres in the Dark, see Genetic Engineering (song).

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Genetic engineering, also called genetic modification, is the direct human manipulation of an organism's genome using modern DNA technology. It involves the introduction of foreign DNA or synthetic genes into the organism of interest. The introduction of new DNA does not require the use of classical genetic methods, however traditional breeding methods are typically used for the propagation of recombinant organisms.

An organism that is generated through the introduction of recombinant DNA is considered to be a genetically modified organism. The first organisms genetically engineered were bacteria in 1973 and then mice in 1974. Insulin-producing bacteria were commercialized in 1982 and genetically modified food has been sold since 1994.

The most common form of genetic engineering involves the insertion of new genetic material at an unspecified location in the host genome. This is accomplished by isolating and copying the genetic material of interest using molecular cloning methods to generate a DNA sequence containing the required genetic elements for expression, and then inserting this construct into the host organism. Other forms of genetic engineering include gene targeting and knocking out specific genes via engineered nucleases such as zinc finger nucleases or engineered homing endonucleases.

Genetic engineering techniques have been applied in numerous fields including research, biotechnology, and medicine. Medicines such as insulin and human growth hormone are now produced in bacteria, experimental mice such as the oncomouse and the knockout mouse are being used for research purposes and insect resistant and/or herbicide tolerant crops have been commercialized. Genetically engineered plants and animals capable of producing biotechnology drugs more cheaply than current methods (called pharming) are also being developed and in 2009 the FDA approved the sale of the pharmaceutical protein antithrombin produced in the milk of genetically engineered goats.

Contents 1 Definition 2 History 3 Process 3.1 Isolating the gene 3.2 Constructs 3.3 Gene targeting 3.4 Transformation 3.5 Selection 3.6 Regeneration 3.7 Confirmation 4 Applications 4.1 Medicine 4.2 Research 4.3 Industrial 4.4 Agriculture 4.5 Other uses 5 Opposition and criticism 6 See also 7 References 8 Further reading 9 External links

Definition

Genetic engineering alters the genetic makeup of an organism using techniques that introduce heritable material prepared outside the organism either directly into the host or into a cell that is then fused or hybridized with the host.^[1] 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 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.^[1] Cloning and stem cell research, although not considered genetic engineering,^[2] are closely related and genetic engineering can be used within them.^[3] 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.^[4]

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.^[5] Genetic engineering can also be used to remove genetic material from the target organism, creating a gene knockout organism.^[6] In Europe genetic modification is synonymous with genetic engineering while within the United States of America it can also refer to conventional breeding methods.^[7] Within the scientific community, the term genetic engineering is not commonly used; more specific terms such as transgenic are preferred.

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,^[8] one year before DNA's role in heredity was confirmed by Alfred Hershey and Martha Chase,^[9] 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.^[10] 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.^[11][12] A year later Rudolf Jaenisch created a transgenic mouse by introducing foreign DNA into its embryo, making it the world’s first transgenic animal.^[13] In 1976 Genentech, the first genetic engineering company was founded by Herbert Boyer and Robert Swanson and a year later and the company produced a human protein (somatostatin) in E.coli. Genentech announced the production of genetically engineered human insulin in 1978.^[14] In 1980, the U.S. Supreme Court in the Diamond v. Chakrabarty case ruled that genetically altered life could be patented.^[15] The insulin produced by bacteria, branded humulin, was approved for release by the Food and Drug Administration in 1982.^[16]

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.^[17] The People’s Republic of China was the first country to commercialize transgenic plants, introducing a virus-resistant tobacco in 1992.^[18] In 1994 Calgene attained approval to commercially release the Flavr Savr tomato, a tomato engineered to have a longer shelf life.^[19] 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.^[20] In 1995, Bt Potato was approved safe by the Environmental Protection Agency, making it the first pesticide producing crop to be approved in the USA.^[21] 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.^[22]

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.^[23][24]

Process

Isolating the gene

Elements of genetic engineering

First, the gene to be inserted into the genetically modified organism must be chosen and isolated. Presently, most genes transferred into plants provide protection against insects or tolerance to herbicides.^[25] In animals the majority of genes used are growth hormone genes.^[26] Once chosen the genes must be isolated. This typically involves multiplying the gene using polymerase chain reaction (PCR). 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. Once isolated, the gene is inserted into a bacterial plasmid.

Constructs

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.^[27]

Gene targeting

Main article: gene targeting

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,^{[28] [29]} engineered homing endonucleases,^{[30] [31]} or nucleases created from TAL effectors.^{[32] [33]} In addition to enhancing gene targeting, engineered nucleases can also be used to introduce mutations at endogenous genes that generate a gene knockout^[34] .^[35]

Transformation

Main article: Transformation (genetics)

A. tumefaciens attaching itself to a carrot cell

About 1% of bacteria are naturally able to take up foreign DNA but it can also be induced in other bacteria.^[36] 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.^[37]

In Agrobacterium-mediated recombination the plasmid construct must also contain T-DNA. Agrobacterium naturally inserts DNA from a tumor inducing plasmid into any susceptible plant's genome it infects, causing crown gall disease. The T-DNA region of this plasmid is responsible for insertion of the DNA. The genes to be inserted are cloned into a binary vector, which contains T-DNA and can be grown in both E. Coli and Agrobacterium. Once the binary vector is constructed the plasmid is transformed into Agrobacterium containing no plasmids and plant cells are infected. The Agrobacterium will then naturally insert the genetic material into the plant cells.^[38]

In biolistics 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.^[39]

Selection

Not all the organism's cells will be transformed with the new genetic material; in most cases a selectable marker is used to differentiate transformed from untransformed cells. If a cell has been successfully transformed with the DNA it will also contain the marker gene. By growing the cells in the presence of an antibiotic or chemical that selects or marks the cells expressing that gene it is possible to separate the transgenic events from the non-transgenic. Another method of screening involves using a DNA probe that will only stick to the inserted gene. A number of strategies have been developed that can remove the selectable marker from the mature transgenic plant.^[40]

Regeneration

As often only a single cell is transformed with genetic material the organism must be regrown 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. 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.

Confirmation

The finding that a recombinant organism contains the inserted genes is not usually sufficient to ensure that the genes will be expressed in an appropriate manner in the intended tissues of the recombinant organism. To examine the presence of the gene, further analysis frequently uses PCR, Southern hybridization, and DNA sequencing, which serve to determine the chromosomal location and copy number of the inserted gene. To examine expression of the trans-gene, an extensive analysis of transcription, RNA processing patterns, and the expression and localization of the protein product(s) is usually necessary, using methods including northern hybridization, quantitative RT-PCR, Western blot, immunofluorescence and phenotypic analysis. When appropriate, the organism's offspring are studied to confirm that the trans-gene and associated phenotype are stably inherited.

Applications

Genetic engineering has applications in medicine, research, industry and agriculture and can be used on a wide range of plants, animals and micro organism.

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.^[41] Vaccination generally involves injecting weak live, killed or inactivated forms of viruses or their toxins into the person being immunized.^[42] Genetically engineered viruses are being developed that can still confer immunity, but lack the infectious sequences.^[43] Mouse hybridomas, cells fused together to create monoclonal antibodies, have been humanised through genetic engineering to create human monoclonal antibodies.^[44]

Genetic engineering is used to create animal models of human diseases. Genetically modified mice are the most common genetically engineered animal model.^[45] They have been used to study and model cancer (the oncomouse), obesity, heart disease, diabetes, arthritis, substance abuse, anxiety, aging and Parkinson disease.^[46] 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.^[47]

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.^[48] Gene therapy has been used to treat patients suffering from immune deficiencies (notably Severe combined immunodeficiency) and trials have been carried out on other genetic disorders.^[49] The success of gene therapy so far has been limited and a patient (Jesse Gelsinger) has died during a clinical trial testing a new treatment.^[50] 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.^[51] The distinction between cure and enhancement can also be difficult to establish.^[52] Transhumanists consider the enhancement of humans desirable.

Research

Knockout mice

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

By engineering genes into bacterial plasmids it is possible to create a biological factory that can produce proteins and enzymes.^[53] Some genes do not work well in bacteria, so yeast, a eukaryote, can also be used.^[54] Bacteria and yeast factories have been 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.^[55] Other applications involving genetically engineered bacteria being investigated involve making the bacteria perform tasks outside their natural cycle, such as cleaning up oil spills, carbon and other toxic waste.^[56]

Agriculture

Bt-toxins present in peanut leaves (bottom image) protect it from extensive damage caused by European corn borer larvae (top image).^[57]

One of the best-known and controversial applications of genetic engineering is the creation of genetically modified food. There are three generations of genetically modified crops.^[58] First generation crops have been commercialized and most provide protection from insects and/or resistance to herbicides. There are also fungal and virus resistant crops developed or in development.^[59][60] They have been developed to make the insect and weed management of crops easier and can indirectly increase crop yield.^[61]

The second generation of genetically modified crops being developed aim to directly improve yield by improving salt, cold or drought tolerance and to increase the nutritional value of the crops.^[62] The third generation consists of pharmaceutical crops, crops that contain edible vaccines and other drugs.^[63] Some agriculturally important animals have been genetically modified with growth hormones to increase their size^[64] while others have been engineered to express drugs and other proteins in their milk.^[65][66][67]

The genetic engineering of agricultural crops can increase the growth rates and resistance to different diseases caused by pathogens and parasites.^[68] 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.^[68]

Ethical and safety concerns have been raised around the use of genetically modified food.^[69] A major safety concern relates to the human health implications of eating genetically modified food, in particular whether toxic or allergic reactions could occur.^[70] Gene flow into related non-transgenic crops, off target effects on beneficial organisms and the impact on biodiversity are important environmental issues.^[71] Ethical concerns involve religious issues, corporate control of the food supply, intellectual property rights and the level of labeling needed on genetically modified products.

Other uses

In materials science, a genetically modified virus has been used to construct a more environmentally friendly lithium-ion battery.^[72][73] Some bacteria have been genetically engineered to create black and white photographs^[74] while others have potential to be used as sensors by expressing a fluorescent protein under certain environmental conditions.^[75] Genetic engineering is also being used to create BioArt^[76] and novelty items such as blue roses,^[77] and glowing fish.^[78]

Opposition and criticism

This section requires expansion.

A 2010 study of Canola found transgenes in 80% of wild (uncultivated or "feral") varieties in North Dakota, meaning 80% of the plants which had established themselves in the area were genetically engineered varieties. The researchers stated that "we found the highest densities of [such transgene-containing] plants near agricultural fields and along major freeways, but we were also finding plants in the middle of nowhere" adding that "over time,..the build-up of different types of herbicide resistance in feral [natural] canola and closely related weeds, like field mustard, could make it more difficult to manage these plants using herbicides."^[79]

See also: Human genetic engineering, GM food controversy, and Genetically modified organism

See also

• Biological engineering
• Genetic engineering in the United States
• Marker assisted selection, a way to select suitable offspring without using genetic engineering
• Paratransgenesis
• Regulation of the release of genetic modified organisms

References

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Further reading

• British Medical Association (1999). The Impact of Genetic Modification on Agriculture, Food and Health. BMJ Books. ISBN 0-7279-1431-6. 
• Donnellan, Craig (2004). Genetic Modification (Issues). Independence Educational Publishers. ISBN 1-86168-288-3. 
• Morgan, Sally (2003). Superfoods: Genetic Modification of Foods (Science at the Edge). Heinemann. ISBN 1-4034-4123-5. 
• Smiley, Sophie (2005). Genetic Modification: Study Guide (Exploring the Issues). Independence Educational Publishers. ISBN 1-86168-307-3. 
• Watson, James D. (2007). Recombinant DNA: Genes and Genomes: A Short Course. San Francisco: W.H. Freeman. ISBN 0-7167-2866-4. 
• Weaver, Sean; Morris, Michael (2003). An Annotated Bibliography of Scientific Publications on the Risks Associated with Genetic Modification. Wellington, N.Z.: Victoria University. http://researcharchive.vuw.ac.nz/handle/10063/3 
• Zaid, A; H.G. Hughes, E. Porceddu, F. Nicholas (2001). Glossary of Biotechnology for Food and Agriculture - A Revised and Augmented Edition of the Glossary of Biotechnology and Genetic Engineering. Available in English, French, Spanish, Arabic. Rome, Italy: FAO. ISBN 92-5-104683-2. http://www.fao.org/biotech/index_glossary.asp. 

External links

Wikimedia Commons has media related to: Genetic engineering

At Wikiversity you can learn more and teach others about Genetic engineering at: The Department of Genetic engineering

• Genetic Engineering on In Our Time at the BBC. (listen now)
• GMO Safety - Information about research projects on the biological safety of genetically modified plants.
• Genetic Engineering A UK site for students, with case studies and ethical responses
• Introduction to Genetic Engineering Covers general information on Genetic Engineering including cloning, stem cells and DNA.
• European Food and Safety Authority
• GMO-compass, news on GMO en EU

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