genetic engineering

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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
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; 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.
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, 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
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; 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.
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), 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.
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). 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. 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

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.

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.

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.

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 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.

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.

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