Genetic engineering

Genetic engineering is the altering of the genetic material of living cells in order to make them capable of producing new substances or performing new functions. When the genetic material within the living cells, i.e. genes are working properly, the human body can develop and function smoothly. However, should a single gene--even a tiny segment of a gene go awry, the effect can bedramatic: deformities, disease, and even death. In the fast 40 years, amazingdiscoveries and development of revolutionary new techniques have allowed scientists to learn a great deal about how genes work and how they are linked todisease.

In the 1950s, largely as the result of the pioneering work of James Watson (1928-) and Francis Crick (1916-), scientists discovered the structure of deoxyribonucleic acid (DNA) molecules and how DNA stores and transmit genetic information. Scientists found that the precise arrangement (or sequence) of nitrogen bases A, C, G. and T on the DNA strand is the recipe that codes for the manufacture of specific chemical compounds, namely proteins. The proteins arethe "work-horses" of the cells and are responsible for carrying out all the functions of the cell. The sequence acts, therefore, as an "instruction manual" that directs all cell functions. If the recipes have extra bases or misspelled bases or if some are deleted, the cell can make a wrong protein or too much or too little of the right one. These mistakes often result in disease. Insome cases, a single misspelled base is sufficient to cause a disease, suchas sickle cell anemia.

Certain practical consequences of that discovery became almost immediately apparent. Suppose that the base sequence T-G-G-C-T-A-C-T on a DNA molecule carries the instruction "make insulin." (The actual sequence for such a message would in reality be very much longer.) DNA in the cells of the islets of Langerhans in the pancreas would normally contain that base sequence since the islets are the regions in which insulin is produced in mammals. But that base sequence carries the same message no matter where it is found. If a way could be found to insert the base sequence into the DNA of bacteria, for example, then those bacteria would be capable of manufacturing insulin.

Although the concept of transferring a base sequence into a bacteria soundedrelatively simple, its actual execution presented a number of difficult technical challenges. The technique required three elements: the gene to be transferred, a host cell in which the gene is to be inserted, and a vector for transferring the gene to the body. Suppose, for example, that one wishes to insert the insulin gene into a bacterial cell. The first step is to obtain a copyof the insulin gene. This copy can be obtained from a natural source (like DNA in islets of Langerhans cells), or it can be manufactured artificially in the laboratory. The second step is to insert the insulin gene into the vector.Viruses, liposomes (hollow spheres of fat molecules formed in solution), andplasmids (circular forms of DNA) are common vectors.

In 1973, an American biochemist Paul Berg often referred to as the father ofgenetic engineering developed a method for joining the DNA of two different organisms, a monkey virus known as SV40 and a second virus known as lambda phage. The accomplishment was significant, but the process was long and laborious. A turning point came later the same year when Stanley Cohn at Stanford andHubert of the University of California at San Francisco discovered an enzymethat greatly increased the efficiency of the Berg process. The technique ofgenetic transfer developed by Berg, Boyer, and Cohen, described below, is fundamentally what is being used in most animal genetic engineering today.

The following events set the stage for Berg, Boyer and Cohen to develop the genetic transfer techniques. In the late 1960s, Werner Arber in Switzerland, and Hamilton Smith, at Johns Hopkins University were studying how some bacteria resist invasion by viruses. They observed that when viruses entered the bacteria, the viral DNA was cut into small pieces and inactivated by certain bacterial enzyme. They called the enzymes that were capable of cutting DNA at specific sequences "restriction endonucleases". Other scientists were quick torecognize that this provided a means of cutting a large DNA molecule into well-defined smaller fragments. These enzymes usually do not cut straight acrossthe two strands of DNA, but in a staggered fashion. Consequently, their cutscreated short single stranded tails on the ends of each fragment, called "sticky ends" or cohesive ends. They were known as cohesive ends because they could form complementary base pairs with any other end produced by the same enzyme. The cohesive ends generated by the restriction endonuclease made it possible to join double helical DNA fragments from different sources by complementary base-pairing. For example, a DNA fragment containing a human gene can bejoined in a test tube to the chromosome of a bacterial virus and a new recombinant DNA molecule could then be introduced into the bacterial cell. Starting with only one such recombinant molecule that infects a single cell, the normal replication mechanism of the virus could produce more than 1012 identicalviral DNA molecules in less than a day, thereby amplifying the amount of theattached human DNA fragment by the same factor. A virus used in this way isknown as a cloning vector.

Plasmids, which are circular extrachromosomal DNA fragments found in bacterial cells, are routinely used for cloning. The plasmid vectors used for gene cloning are derived from large plasmids that are found naturally in bacteria, yeast, and mammalian cells. For use as cloning vectors, the plasmids are cut with a restriction endonuclease to create linear DNA molecules. The gene, which is to cloned, is cut with the same restriction endonuclease and the fragments are added to the cut plasmids and annealed to form recombinant DNA circles. These recombinant DNA molecules containing foreign DNA inserts are sealed with an enzyme called "ligase" which fills the gap and completes the restoration process to form intact DNA circles.

The recombinant DNA circles are then introduced into cells (usually bacterialor yeast cells) that have been made transiently permeable to the DNA. Thesecells are said to be transfected by plasmids. As these cells grow and divide,the recombinant DNA molecules replicate to produce an enormous number of copies of DNA circles containing the foreign DNA. Because a single bacterium grows rapidly, producing more than a billion copies of itself in 15 hours, largequantities of a specific DNA sequence could be produced in this manner--called cloning. This method is sometimes referred to as gene splicing.

Since genes from two different sources have been combined with each other, the technique is also called recombinant DNA (rDNA) research. A variety of expression vectors such as plasmids and even certain viruses could be used for this purpose. These vectors are called expression vectors and are engineered tofunction in the type of cell in which the protein is to be made. By means ofsuch genetic engineering, bacteria, yeast or even mammalian cells can be induced to make large quantities of useful proteins--such as human growth hormone, interferon, and viral antigens for vaccines. Bacterial cells with plasmidor viral vectors that have been engineered in this way are especially adept at protein production and can produce more than 10% of the total protein.

The possible applications of genetic engineering are nearly limitless. For example, rDNA methods now make it possible to produce a number of natural products that were previously available in only very limited amounts. Until the 1980s, for example, the only supply of insulin available to diabetics was animals slaughtered for meat or other purposes. That supply was never adequate totreat all diabetics at moderate cost. In 1982, however, the United States Food and Drug Administration approved insulin produced by genetically altered organisms, the first such product to become available. Since 1982, a number ofadditional products, including human growth hormone, alpha interferon, interleukin-2, erythropoietin, tumor necrosis factor, and tissue plasminogen activator have been produced by rDNA techniques. In addition to the production of insulin, this technique has been used to create recombinant factor VIII for the treatment of hemophilia, a hereditary blood defect that inhibits blood clotting and makes it difficult for the body to naturally control bleeding. Thisgenetically engineered blood factor protein can help induce clotting. Available since 1993, factor VIII can, for example, be produced in hamster cell lines using Bovine serum proteins. It is considered safer than similar blood-derived factors, which have the potential to pass on blood viruses, such as AIDSor hepatitis. Several other similar blood factor products are under development.

The potential commercial value of genetically engineered products was not lost on entrepreneurs in the 1970s. In many cases, the founders of the first genetic engineering firms were scientists themselves, often those involved in basic research in the field. Boyer, for example, joined with venture capitalistRobert Swanson in 1976 to form Genentech (Genetic Engineering Technology). Other early firms like Cetus, Biogen, and Genex were formed similarly throughthe collaboration of scientists and business people. The structure of geneticengineering (or, more generally, biotechnology) firms has been a source of controversy, with concerns about individual scientists making a profit by opening their own companies that are based on research carried out at public universities and paid for with federal funds. By the early 1990s, working relationships had, in many cases, been formalized among universities, individual researchers, and the corporations they establish. But not everyone is satisfiedthat the ethical issues involved in such arrangements are settled.

One of the most exciting potential applications of genetic engineering involves the treatment of genetic disorders. Medical scientists now know of more 3,000 disorders that arise because of errors in an individual's DNA and are continuously finding new links among genes and diseases. Conditions such as sickle-cell anemia, Tay-Sachs disease, Duchenne muscular dystrophy, Huntington'schorea, cystic fibrosis, and Lesch-Nyhan syndrome are the result of the loss,mistaken insertion, or change of a single nitrogen base in a DNA molecule. Genetic engineering makes it possible for scientists to provide individuals who lack a certain gene with correct copies of that gene. If and when that correct gene begins to function, the genetic disorder may be cured. This procedure is known as human gene therapy.

The first approved trials of gene therapy with human patients were begun in 1989. One of the most promising sets of experiments involved a condition knownas severe combined immune deficiency (SCID) or ADA deficiency. Children bornwith this disorder have no immune system because of the lack of a single gene. In 1990, a research team at the National Institutes of Health led by W. French Anderson attempted gene therapy with a four-year old patient with SCID.The patient received about a billion cells containing a genetically engineered copy of the ADA gene that the child's body lacked. Human gene therapy is the source of great controversy among scientists and non-scientists alike. However, few individuals would say that the technique should never be used, especially for battling life threatening diseases, like AIDS and cancer. But manycritics worry about where gene therapy might lead, such as genetically engineered humans.

Genetic engineering also promises a revolution in agriculture. Recombinant DNA techniques make it possible to produce plants that are resistant to herbicides, that will survive freezing temperatures, that will take longer to ripen,that will convert atmospheric nitrogen to a form they can use, that will manufacture their own resistance to pests, and so on. Scientists have tested a multitude of plants engineered to have special properties such as these. In 1994, a tomato was the first genetically engineered food to appear in Americansupermarkets. The genetically engineered tomato was created with an "antisense" gene that allows the tomato to ripen on the vine but remain firm for shipping.

As with every other aspect of genetic engineering, however, these advances have been controversial. The development of herbicide-resistant plants, for example, only means that farmers will use still larger quantities of herbicides,critics say, not an especially desirable trend. How sure can we be, others ask, about the potential risk to the environment posed by the introduction of"unnatural" engineered plants? For example, in the case of the tomato, some people are concerned genes from the plant could spread into soil bacteria andthen infect humans or animals. Many other applications of genetic engineeringhave already been developed or are likely to be realized in the future. In every case, however, the glowing promises of each new technique is somewhat tempered by the social, economic and ethical questions it raises.

Genetically engineered clones (exact genetic copies of an individual), for example, became a major issue in 1997 when scientists from Scotland announced that they had successfully created the first clone of an adult mammal. The genes used to clone Dolly the sheep came from the frozen mammary tissue of a six-year-old dead sheep. The cloning was accomplished by taking the cell nucleusfrom a Finn Dorset sheep and then substituting it with the egg from the deadPoll Dorset sheep. The nucleus with the egg was then implanted into a thirdScottish Blackface sheep. Because of the three different breeds used, the researchers had ready evidence that Dolly was truly a clone. While Dolly was a major advance scientifically, she became more famous among the public becauseof the ethical furor that has surrounded her "creation." Using genetic engineering to clone humans could be abused to create certain "types" of people, bringing forth the specter of humans as objects instead of individuals. Both the United States and Great Britain have banned human cloning. Still, the cloning of animals has many potential benefits, including raising animals that could be genetically engineered to produce more meat or, perhaps, to produce a human gene protein that could then be used in gene therapy.

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