Clone, also spelled Clon, is a population of genetically identical cells or organisms that are derived originally from a single original cell or organism by asexual methods.

Cloning is fundamental to most living things, since the body cells of plants and animals are clones ultimately derived from the mitosis of a single fertilized egg.

More narrowly, a clone can be defined as an individual organism that was grown from a single body cell of its parent and that is genetically identical to it.

Plants that are able to propagate by asexual means produce genetically identical plants that are clones.

Cloning has been commonplace in horticulture since ancient times; many varieties of plants are cloned simply by obtaining cuttings of their leaves, stems, or roots and replanting them.

A vast array of fruit and nut tree varieties and innumerable ornamental plants represent clones.

The body cells of adult animals and humans can be routinely cloned in the laboratory.

Adults cells of various tissues, such as muscle cells, that are removed from the donor animal and maintained on a culture medium while receiving nutrients manage not only to survive but to go on dividing, producing colonies of identical descendants.

By the 1950s scientists were able to clone frogs, producing identical individuals that carry the genetic characteristics of only a single parent.

The technique used in the cloning of frogs consists of transplanting frog DNA, contained in the nucleus of a body cell, into an egg cell whose own genetic material has been removed.

The fused cells then begin to grow and divide, just like a normal fertilized egg, to form an embryo.

Mice were first successfully cloned in the 1980s, using a procedure in which the nucleus from a body cell of a mouse embryo is removed from the uterus of a pregnant mouse and transplanted into a recently fertilized egg (from another mouse) whose genetic contents have been evacuated.

The cell is cultured artificially until it divides and becomes an embryo.

The embryo, which is composed entirely of cells derived from the single implanted nucleus, is artificially implanted into the uterus of another mouse that brings it to term.

Cloning a new animal from the cells of an adult (as opposed to those of an embryo) is considerably more difficult, however. Almost all of an animal's cells contain the genetic information needed to reproduce a copy of the organism.

But as cells differentiate into the various tissues and organs of a developing animal, they express only that genetic information needed to reproduce their own cell type. This tended to restrict animal cloning to the use of embryonic cells, which have not yet differentiated into blood, skin, bone, or other specialized cells, and which can more easily be induced to grow into an entire organism.

The first success in cloning an adult mammal was achieved by a team of British researchers led by Ian Wilmut at the Roslin Institute in Edinburgh, Scot., in 1996.

After having already produced clones from sheep embryos, they were able to produce a lamb using DNA from an adult sheep.

In their procedure, the nucleus of a cell from the mammary gland of an adult sheep was implanted in another sheep's unfertilized egg whose nucleus had been removed.

The key to the new procedure is to synchronize the cell cycle--i.e., the ordered sequence of events that occur in a cell in preparation for division--of the mammary cell with that of the egg. To achieve this, before implantation the mammary cell is deprived of nutrients; this stops its cell cycle, thus preventing it from dividing.

The nucleus is then implanted into the recipient egg and fused to it, and an electrical current is applied to simulate the burst of energy that occurs during fertilization. The egg begins dividing normally and becomes an embryo, which is implanted into another ewe. The lamb that is born is a clone of the donor of the original mammary cell.

The practical applications of cloning are economically promising but philosophically unsettling. Animal breeders would welcome the chance to clone top-quality livestock. Genetically engineered animals could be cloned in large numbers to increase the production of drugs or human proteins that are useful in fighting disease. Clones are also highly useful in biological research because of their genetic uniformity.

The cloning of human beings is a subject fraught with ethical and moral controversy. If cloning can ensure the infinite replication of specific genetic traits, a judgment would need to be made as to which traits are desirable and therefore worthy of perpetuation. The persons empowered to exercise such judgment would be in a position to change the course of human development.

The use of recombinant DNA technology to manipulate and change genes is sometimes called gene cloning. See genetic engineering.

Since the 1970s, biologists have made major advances in understanding the molecular nature of genes and their functioning through the use of the powerful experimental techniques of recombinant DNA.

The term recombinant DNA literally means the joining or recombining of two pieces of DNA from two different species. Recombinant DNA techniques allow an investigator to biologically purify (clone) a gene from one species by inserting it into the DNA of another species, where it is replicated along with the host DNA.

Actually, the term includes a variety of molecular manoeuvres, including cleaving DNA by microbial enzymes called endonucleases, splicing or recombining fragments of DNA, inserting eucaryotic DNA into bacteria so that large quantities of the foreign genetic material can be produced, determining the nucleotide sequence of a segment of DNA, and even chemically synthesizing DNA.

Gene cloning ranks as one of the most significant accomplishments involving recombinant DNA. This procedure has enabled researchers to use E. coli to produce virtually limitless copies of donor genes from other organisms, including human beings.

To perform gene cloning, researchers first use a class of bacterial enzymes called restriction endonucleases to remove from the donor cell a fragment of double-stranded DNA that contains the genes of interest. Restriction endonucleases can be thought of as "biological scissors"; each of these enzymes cleaves DNA at a specific site defined by a sequence of four or more nucleotides.

Once the desired DNA fragment has been removed from the donor cell, it must somehow be inserted into the bacterial cell. This is usually done by first inserting the donor DNA into a plasmid, one of the small, circular pieces of DNA that are found in E. coli and many other bacteria.

Plasmids generally remain separate from the bacterial chromosome (although some plasmids do occasionally become incorporated into the chromosome), but they carry genes that can be expressed in the bacterium. Furthermore, plasmids generally replicate and are passed on to daughter cells along with the chromosome.

By treating a plasmid with the same restriction endonuclease that was used to cleave the donor DNA, it is possible to incorporate the foreign DNA fragment into the plasmid ring. This can occur because the restriction enzyme cleaves double-stranded DNA in such a way as to leave chemically "sticky" end pieces. It is thus possible for the sticky-ended fragment of foreign DNA to attach to the complementary sticky ends of the cut-open plasmid ring. This laboratory procedure, called "gene splicing," is the major operation of recombinant DNA technology.

The molecular biologist then uses the plasmids as vectors to carry the foreign gene into bacteria. This is accomplished by exposing bacteria to the plasmids. Plasmids are highly infective, and so many of the bacteria will take up the particles; to insure maximum uptake the bacteria are often treated with calcium salts, which makes their membranes more permeable.

The incorporation of the plasmids into the bacterial cells marks the transfer of the genes of one species into the genome of another. Alternatively, bacteriophages are sometimes used as vectors to carry the foreign DNA into the bacteria.

As a result of the high infectivity of plasmids and the rapid growth of E. coli, investigators can quickly culture large numbers of bacteria, many of which will have incorporated the foreign (often human) DNA. (As many as 1 109 bacteria can grow in one millilitre of medium overnight.) Researchers can select the bacteria that contain the foreign DNA by attaching to the fragment of DNA a gene that confers resistance to an antibiotic such as tetracycline.

By treating the culture with tetracycline, all bacteria that have not incorporated the gene for resistance will be killed. The remaining cells can be grown in enormous numbers, most of which will contain the cloned fragment of foreign DNA.

The cloned DNA can be removed from the bacterial culture as follows. First, the bacteria are broken apart and the DNA content is separated by centrifugation. The DNA fraction is then heated, which causes the double-stranded molecules to separate into single strands. Upon cooling, each single strand will reanneal, or hybridize, to another single strand to which it is complementary (adenine opposite thymine, cytosine opposite guanine). This form of molecular hybridization has made possible the use of the complementary DNA (cDNA) as a probe for picking out the desired gene.

Investigators also use DNA to pick out a specific gene from a large piece of genomic DNA. In some cases where a cell makes large amounts of specific mRNA (such as globin mRNA in human red cells), the extracted mRNA can be treated with reverse transcriptase to produce cDNA. When labelled with a radioisotope, this then becomes a cDNA probe for the human globin gene. The technique for isolating and hybridizing the fragment of interest is called "southern blot" analysis.

The huge number of copies attained by gene cloning enables researchers to analyze the cloned DNA exhaustively, down to its nucleotide sequence. Nucleotide sequencing is accomplished by performing a series of biochemical manoeuvres on small, endonuclease-produced fragments (oligonucleotides) and then placing them in the correct order. Remarkably, molecular biologists have automated the entire procedure so that a "gene machine" can determine the nucleotide sequence of a gene in a relatively short time. In fact, if the amino-acid sequence of a protein is known, researchers can formulate the nucleotide sequence that produced it and then synthesize the gene. This has been done for insulin.

A given restriction endonuclease can produce a very large number of discrete DNA fragments, which can be inserted into vector DNA incised by the same endonuclease. Researchers can clone the fragments as described above to produce a so-called library of genomic DNA. This library can be used to study the natural gene whenever a new probe is obtained.

A given restriction enzyme generally will produce fragments that are the same for all individuals. However, different people occasionally vary in the size of specific fragments. This is due to the fact that in any string of several hundred bases in human DNA there occur single base changes, usually harmless substitutions that either change or remove the enzyme site.

These fragment variations, known as restriction-fragment-length polymorphisms, are inherited and hence form genetic markers that can be used to trace mutant genes to which they are linked. If the fragments are separated by agarose gel electrophoresis and overlaid with a radioactively labelled cDNA probe, only the fragment whose DNA is complementary to that of the probe will hybridize with it.

This hybridization can be detected by exposing the DNA fragments to photographic film; the resultant image is called an autoradiograph. When restriction-fragment-length polymorphisms are present, different-sized fragments will hybridize with the same probe. Linkage studies between a disease-producing mutant gene and a polymorphism will locate the gene to either the polymorphic or "wild-type" (i.e., normal) fragment.

If large family studies show that the gene is linked closely enough to the fragment so that recombination is rare, this technique can be used for diagnosing the presence of a genetic disease for which the biochemical defect is unknown (see below).

One further result of the ability to analyze gene structure at the molecular level has been the discovery of its remarkable plasticity. Investigators have found sequences of nucleotides that have the capacity to move from one position on the chromosome to another, often carrying neighbouring sequences with them and thus rearranging the DNA.

These "jumping genes" - known as transposable elements, or transposons--have been found in both procaryotes and eucaryotes. In the higher mammals, including humans, they are the source of the tremendous diversity necessary for antibody production by the immune system.

It is also possible that some forms of cancer may develop as a result of these rearrangements.

In addition to producing copies of genes for molecular analysis and for use in medical diagnosis, recombinant DNA procedures have been used to convert bacteria into "factories" for the synthesis of foreign proteins. This is a tricky operation, for not only must the foreign DNA be inserted into the host bacterium, but it also must be incorporated into an operon so that its product will be expressed. Despite the technical difficulties, investigators have achieved the expression of foreign genes within E. coli.

This fact has tremendous potential in medicine, as the "engineered" bacteria can be used to produce therapeutically valuable human proteins. Insulin, growth hormone, and antihemophilic globulin (the clotting factor missing in persons with hemophilia) are three such proteins that have been commercially "manufactured" via recombinant DNA in E. coli.

As a result of this "engineering," the host bacterium has been provided with new genetic properties. Both the scientific and lay communities have expressed concern over the creation of microorganisms with new genetic properties. Perhaps this genetic tailoring of infectious agents like E. coli could visit new and devastating epidemics on the population or could introduce cancer-causing genes into infected people.

In the United States, federal agencies, with the assistance of molecular biologists, have laid down stringent guidelines to ensure the control of microorganisms containing the recombinant plasmids. The most effective measure has been the requirement to use strains of E. coli that have been modified so that they can survive in the laboratory but not in nature (and hence are not infectious).

In addition, the guidelines require a physical containment system that securely seals off the laboratory, thereby preventing the escape of the bacteria from the facility. Molecular biologists have also called attention to the fact that recombinant processes are constantly occurring in nature, albeit at a slower rate.

Other forms of genetic engineering

The techniques discussed so far, all of which are outgrowths of somatic cell genetics, have made it possible to manipulate the genetic systems of a variety of organisms. The popular press has dubbed this research "genetic engineering" and has implied that it is somehow dangerous, unnatural, and immoral, especially when human chromosomes are manipulated.

In one sense, of course, the selective breeding of desirable traits in both plants and animals has been practiced since ancient times and is a form of genetic manipulation. For this discussion, however, genetic engineering will be limited to the deliberate laboratory manipulation of the cellular or nucleic acid constitution of an organism so as to produce a specific genetic change that will persist as the cell, or cells, multiply.

The genetic engineering made possible by these techniques has caused people to consider social, ethical, and philosophical issues raised by the production of new forms of life. The U.S. Supreme Court has stated that the production of a new bacterium (E. coli) "with markedly different characteristics from any found in nature" may be patented by the scientist who makes it. Many have found this an objectionable form of "playing God," which diminishes the mystery of life.

Others claim that this new biotechnology developed out of human efforts to understand life and does not in the slightest affect its mystery. The nightmare of "mad geneticists" using these techniques to permanently alter the phenotype of the human species or to "clone" humans to genetic specifications may be possible in the distant future. To guard against such abuses requires careful regulation of various types of research on human embryos.

In addition to recombinant DNA, other forms of genetic manipulation that will increase knowledge of developmental processes and eventually may be of assistance in the replacement of mutant genes by normal genes include gene transfer, embryo transfer, and nuclear transplantation.

Gene transfer ranks as one of the most promising methods for increasing understanding of developmental genetics. In gene transfer, molecular biologists isolate segments of DNA containing a gene or genes of interest and then incorporate them into the DNA of somatic cells in culture.

Investigators transfer the genes with the assistance of calcium ion by a process called transfection; alternatively, they can insert the genes by the delicate procedure of microinjection. These genes replicate with the cells and on occasion will produce their specific protein products.

One mammal to which gene transfer has been successfully applied is the mouse. Researchers have microinjected foreign genes into fertilized mouse eggs and then transferred the surviving embryos into the uterus of an appropriately prepared (pseudopregnant) female mouse, where implantation and gestation occur. The mice born from these experiments have produced the foreign gene product and have passed the new gene on to their offspring.

It should be emphasized that this procedure has the capability not only of counteracting the effects of a defective gene in the mouse but also of passing on the new gene to future generations, thus effecting a true genetic cure. Similar gene-transfer procedures might possibly be effective in treating genetic diseases in humans.

Embryo transfer refers to the process of transferring a pre-implantation embryo from the reproductive tract of one female to that of another, where it often implants and goes through a normal gestation. In an elaboration of this method in the 1960s, Beatrice Mintz and her coworkers disaggregated the cells from early embryos of two genetically different pure lines of mice, mixed them together, and then reimplanted the newly formed embryos into the uterus of a foster mother. The embryos developed and were born as tetraparental (allophenic) mice.

In nuclear transplantation, molecular biologists transfer the entire nucleus of one cell into a second, enucleated cell (i.e., a cell that has had its nucleus removed). This work has had its broadest application in the study of cell differentiation in higher animals. By transplanting the nucleus from a differentiated cell into a less specialized one, investigators have sought to answer some of the riddles of differentiation.

One of the landmark experiments in nuclear transplantation was performed by the British molecular biologist John B. Gurdon. Gurdon transplanted a mature nucleus from an intestinal cell of a tadpole into an enucleated frog's egg which subsequently developed into a normal, adult frog. Gurdon thus demonstrated that a highly differentiated intestinal cell nucleus, with only intestinal cell genes functioning, could undifferentiate in the environment of the enucleated egg cell and could reactivate those genes necessary to create an entire frog.

The frog that was produced was a "clone" in the sense that the entire genome of the donor tadpole was present in all the cells of the newly formed frog. Even such a frog, however, is not an exact replica of the donor because of the multiple levels at which gene expression is affected by changing environments.

Cells from an adult frog, rather than a tadpole, were not successful in this experiment. Although it is not impossible that humans could be cloned in this way in the future, such cloning would be extremely difficult and of questionable use, certainly for large numbers.

The moral dilemmas raised by this type of genetic engineering would be most serious.

- Encyclopedia Britannica