genetic engineering

genetic engineering,

the use of various methods to manipulate the DNA (genetic material) of cells to change hereditary traits or produce biological products. The techniques include the use of hybridomas (hybrids of rapidly multiplying cancer cells and of cells that make a desired antibody) to make monoclonal antibodiesmonoclonal antibody,
an antibody that is mass produced in the laboratory from a single clone and that recognizes only one antigen. Monoclonal antibodies are typically made by fusing a normally short-lived, antibody-producing B cell (see immunity) to a fast-growing cell, such as
..... Click the link for more information. ; gene splicing or recombinant DNA, in which the DNA of a desired gene is inserted into the DNA of a bacterium, which then reproduces itself, yielding more of the desired gene; polymerase chain reactionpolymerase chain reaction
(PCR), laboratory process in which a particular DNA segment from a mixture of DNA chains is rapidly replicated, producing a large, readily analyzed sample of a piece of DNA; the process is sometimes called DNA amplification.
..... Click the link for more information. , which makes perfect copies of DNA fragments and is used in DNA fingerprintingDNA fingerprinting
or DNA profiling,
any of several similar techniques for analyzing and comparing DNA from separate sources, used especially in law enforcement to identify suspects from hair, blood, semen, or other biological materials found at the scene of a violent
..... Click the link for more information. ; and any of several genome-editing techniques involving nucleases (enzymes that cut the bonds that join nucleotidesnucleotide
, organic substance that serves as a monomer in forming nucleic acids. Nucleotides consist of either a purine or a pyrimidine base, a ribose or deoxyribose, and a phosphate group. Adenosine triphosphate serves as the principle energy carrier for the cell's reactions.
..... Click the link for more information. ), especially the CRISPR-Cas9 system, which can more easily edit or engineer genes and also has been used experimentally to activate and deactivate genes.

Genetically engineered products include bacteria designed to break down oil slicks and industrial waste products, drugs (human and bovine growth hormones, human insulin, interferon), and plants that are resistant to diseases, insects, and herbicides, that yield fruits or vegetables with desired qualities, or that produce toxins that act as pesticides. Genetic engineering techniques have also been used in the direct genetic alteration of livestock and laboratory animals (see pharmingpharming
, the use of genetically altered livestock, such as cows, goats, pigs, and chickens, to produce medically useful products. In pharming, researchers first create hybrid genes using animal DNA and the human or other gene that makes a desired substance, such as a hormone.
..... Click the link for more information. ). In 2014 scientists at the Scripps Research Institute created genetically engineered Escherichia coli bacteria that included a pair of synthetic nucleotides, or DNA bases, in its genetic code. A Chinese scientist claimed in 2018 to have genetically edited human embryos prior to implantation using the CRISPR technique to increase resistance to HIV infection; the claim led to an international outcry denouncing his work as unethical human experimentation, and China later formally banned such work. Genetically engineered products usually require the approval of at least one U.S. government agency, such as the Dept. of Agriculture, the Food and Drug Administration, or the Environmental Protection Agency.

Because genetic engineering involves techniques used to obtain patents on human genes and to create patentable living organisms, it has raised many legal and ethical issues. The safety of releasing into the environment genetically altered organisms that might disrupt ecosystems has also been questioned. The discovery in 2001 of genetically engineered DNA in native Mexican corn varieties made concerns of genetic pollution actual, and led some scientists to worry that the spread of transgenes through cross-pollination could lead to a reduction in genetic diversity in important crops. Transgenic rape (canola) plants also have been found in the wild in several countries. Imports of genetically modified corn, soybeans, and other crops have been curtailed or limited in some countries, and the vast majority of such crops are grown in just a handful of nations. The Cartagena Protocol on Biosafety, which has been signed by more than 100 nations and took effect in Sept., 2003, requires detailed information on whether and how imported seeds, plants, animals, other organisms, and the like are genetically modified and permits a nation to bar those imports, but a 2006 World Trade Organization decision treated the banning of genetically modified crops as a form of protectionism. The United States is not party to the 2003 treaty.

Genetic engineering

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 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 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 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.

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 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 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 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 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 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 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 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 Breeding (plant), Gene, Gene action, Somatic cell genetics

genetic engineering

the process of artificially combining genetic material to produce new varieties of plants and animals or new medical treatments. The process is controversial, given that there may be unknown ecological implications. The uncertainty involved is characteristic of the new ‘manufactured’ risks seen as characterizing the RISK SOCIETY. see also HUMAN GENOME PROJECT.

genetic engineering

[jə¦ned·ik en·jə′nir·iŋ] (genetics) The intentional production of new genes and alteration of genomes by the substitution or addition of new genetic material. Also known as biogenetics.

Genetic engineering

Basic techniques

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. Genetic engineering methods 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

alteration of the DNA of a cell for purposes of research, as a means of manufacturing animal proteins, correcting genetic defects, or making improvements to plants and animals bred by man

Genetic Engineering

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 as 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 out-side 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. Prior to this development, cloning had been successful only with immature cells, not those from an adult animal. The breakthrough raised the prospect of human cloning and prompted an international debate regarding the ethical and legal implications of cloning.

Since the cloning of the sheep, nicknamed "Dolly," scientists have found the process of cloning to be more difficult than expected. Since Dolly, scientists have cloned such animals as cows, pigs, monkeys, cats, and even rare and endangered animals. The process of cloning is complex, involving the replacement of the nucleus of an egg cell with the nucleus of a cell from the subject that will be cloned. This process is meticulous, and the failure rate is high.

In November 2001, scientists first successfully inserted the DNA from one human cell into another human egg. Although the eggs began to replicate, they died shortly after the procedure. Human cloning has caused the most intense debate on the issue, with the debate focusing upon scientific, moral, and religious concerns over this possibility. Scientists do not expect that human cloning will be possible for several years.

Evidence suggests that cloned animals have experienced significant health problems, leading to concerns about the vitality of the entire process. Cloned animals tend to be larger at birth, which could cause problems for the female animals giving birth to them. The cloned organisms also tend to become obese at middle age, at least in the case of experimental cloned mice. Moreover, evidence suggests that cloned animals have died because they do not have sufficient Immunity defenses to fight disease.

Dolly lived for six years before dying in February 2003, which is about half of the normal life expectancy of a sheep. Proponents of the cloning experiments suggest that cloning opens a number of possibilities in scientific research, including the nature of certain diseases and the development of genetically-enhanced medications. Scientists have also successfully cloned endangered animals. In 2001, an Italian group cloned an endangered form of sheep, called the European mouflon. About a year and a half earlier, an American company, Advanced Cell Technology, tried unsuccessfully to clone a rare Asian ox. The cloning was initially successful, but the animal died of dysentery 48 hours after birth.

In 2000, a group of 138 countries, including the United States, approved the Cartagena Protocol on Biosafety Environment. International concerns over the handling of genetically modified organisms (GMOs) prompted the passage of the protocol. It governs such issues as the safe transfer, handling, use, and disposals of GMOs among member countries.

Cross-references

Genetic Screening.

genetic engineering

genetic engineering

genetic engineering

genet′ic engineer′ing

genetic engineering

gene

genetic engineering

genetic engineering,

Genetic engineering

Basic techniques

Isolation of passenger DNA

Joining DNA molecules

Transformation

Cloning vectors

Prokaryotes and lower eukaryotes

Animal cells

Plant cells

Applications

genetic engineering

genetic engineering

Genetic engineering

Basic techniques

Applications

genetic engineering

genetic engineering

ge·net·ic en·gi·neer·ing

genetic engineering

biotechnology

genetic engineering

genetic engineering

genetic engineering

Genetic engineering

Genetic Engineering

Genetic Engineering

International Codes and Ethical Issues for Society

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

Significant State Laws

Recent Developments

Further readings

Cross-references

genetic engineering

Synonyms for genetic engineering

noun the technology of preparing recombinant DNA in vitro by cutting up DNA molecules and splicing together fragments from more than one organism

Synonyms

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