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Cloning
(genetics) The process of asexual reproduction observed, for example, in bacteria and other unicellular micro-organisms which divide by simple fission, so that the daughter cells are genetically identical to each other and to the parent (except when mutation occurs). In higher organisms, genetically identical individuals may be produced by cloning. A body (somatic) cell is taken from an embryo in an early stage of development or from an adult, the nucleus transferred to an unfertilized ovum from which the nucleus has been removed, and the product grown in culture; daughter cells from the earliest divisions are removed, and grown in culture or implanted into host mothers to give genetically identical offspring. The successful cloning of a sheep (named Dolly, 1996–2003) was reported by scientists from the Roslin Institute, Edinburgh, UK, in February 1997. There is considerable potential application in animal rearing, but its application to humans is extremely unlikely (except in some rare instances of in vitro fertilization). The term molecular cloning is used in recombinant DNA technology, where a section of foreign DNA is inserted into an artificial bacterial chromosome (plasmid) and divides with it, thus ‘cloning’ the DNA.
In 1998, a Council of Europe protocol banning the cloning of human beings was signed in Paris by 19 states - the first international treaty on the issue. However, in 1999, predictions were being made about the application of the technique in other areas, such as bone-marrow grafting in leukaemia, and transplant medicine in general, and the controversy surrounding the ethics of human cloning continued to exercise professional and public opinion. In 2004, Newcastle University received Britain's first licence to clone human embryos. In early 2006, Dr Hwang Woo-suk, a South Korean scientist working at Seoul National University, was disgraced when he admitted that his recently published research on human stem cells had been fabricated.
This page discusses genetic cloning. For the computer-related use of the term, see Disk cloning.
Cloning is the process of creating an identical copy of an original organism or thing. A cloning in the biological sense, therefore, is a molecule, single cell (like bacteria, lymphocytes etc.) or multi-cellular organism that has been directly copied from and is therefore genetically identical to another living organism. Sometimes this term can refer to "natural" clones made either when an organism asexual reproduced by chance (as with identical twins), but in common parlance, a clone is an identical copy created intentionally.
The term clone is derived from κλων, the Greek word for "twig". In horticulture, the spelling clon was used until the twentieth century; the final e came into use to indicate the vowel is a "long o" instead of a "short o". Since the term entered the popular lexicon in a more general context, the spelling clone has been used exclusively.
Molecular cloning
Molecular cloning refers to the procedure isolating a DNA sequence of interest and obtaining multiple copies of it in an organism. Cloning is frequently employed to amplify DNA fragments containing genes, an essential step in their subsequent analysis. Frequently, the term cloning is misleadingly used to refer to the identification of the chromosomal location of a gene associated with a particular phenotype of interest. In practice, localisation of the gene does not always enable one to amplify the relevant genomic sequence.
Cloning of any DNA sequence involves the following four steps: amplification, ligation, transfection, and screening/selection. Initially, the DNA fragment of interest needs to be amplified (many copies need to be produced). Amplification is commonly achieved by means of PCR. Subsequently, a ligation procedure is employed whereby the amplified fragment is inserted into a vector. The vector (which is frequently circular) is linearised by means of restriction enzymes, and incubated with the fragment of interest under appropriate conditions that allow for ligation. The yield of the ligation is typically low and depends on the procedure employed. Following ligation the vector with the insert of interest is transfected to cells. Most commonly electroporation is employed, although a number of alternative techniques are available, such as chemical sensitivation of cells. Finally, the transfected cells are cultured. As the aforementioned procedures are of particularly low yield, there is the need to identify the cell colonies that have been transfected with the construct of interest containing the desired insertion sequence. Modern cloning vectors include selectable antibiotic resistance markers, which allow only for cells in which the vector has been transfected to grow. However this selection step does not guarantee that the DNA insert is present in the vector. Further investigation of the resulting colonies is required to confirm that cloning was successful. This can be accomplished by means of blue/white screening (α-factor complimentation) on X-gal medium and/or PCR, possibly followed by DNA sequencing.
Genetic cloning
Cloning a cell means to derive a (clonal) population of cells from a single cell. This is an important in vitro procedure when the expansion of a single cell with certain characteristics is desired, for example in the production of gene-targeted ES cells. Most individuals began as a single cell and are therefore the result of clonal expansion in vivo.
Organism
Cloning means to create a new organism with the same genetic information as a cell from an existing one(identical). It is an asexual method of reproduction, where fertilization or inter-gamete contact does not take place. Asexual reproduction (also known as agamogenesis) is a form of reproduction which does not involve meiosis, gamete formation, or fertilization. In laymen's terms, there is only one "parent" involved. This form of reproduction is common among simple organisms such as amoeba and other single-celled organisms, although most plants reproduce asexually as well (see vegetative reproduction).
Horticultural
The term clone is used in horticulture to mean all descendants of a single plant, produced by vegetative reproduction or apomixis. Many horticultural plant cultivars are clones, having been derived from a single individual, multiplied by some process other than sexual reproduction. As an example, some European cultivars of grapes represent clones that have been propagated for over two millennia. Other examples are potato and banana. Grafting can be regarded as cloning, since all the shoots and branches coming from the graft are genetically a clone of a single individual, although the root systems may be genetically genuine examples of cloning in the broader biological sense, as they create genetically identical organisms by biological means, but this particular kind of cloning has not come under ethical scrutiny and is generally treated as an entirely different kind of operation.
Many trees, shrubs, vines, ferns and other herbaceous perennials form clonal colonies. Parts of a large clonal colony often become detached from the parent, termed fragmentation, to form separate individuals. Some plants also form seeds asexually, termed apomixis, e.g. dandelion.
Animals
Cloning exists in nature in some animal species and is referred to as parthenogenesis. An example is the "Little Fire Ant" (Wasmannia auropunctata), which is native to Central and South America but has spread throughout many tropical environments.
Reproductive Cloning
Reproductive cloning is a technology used to generate an animal that has the same nuclear DNA as another currently or previously existing animal. Dolly the sheep, was created by reproductive cloning technology. In a process called "somatic cell nuclear transfer" (SCNT), scientists transfer genetic material from the nucleus of a donor adult cell to an egg whose nucleus, and thus its genetic material, has been removed. The reconstructed egg containing the DNA from a donor cell must be treated with chemicals or electric current in order to stimulate cell division. Once the cloned embryo reaches a suitable stage, it is transferred to the uterus of a female host where it continues to develop until birth.
Dolly or any other animal created using nuclear transfer technology is not truly an identical clone of the donor animal. Only the clone's chromosomal or nuclear DNA is the same as the donor. Some of the clone's genetic materials come from the mitochondria in the cytoplasm of the enucleated egg. Mitochondria, which are organelles that serve as power sources to the cell, contain their own short segments of DNA, although this is only 0.01% of the total DNA. Acquired mutations in mitochondrial DNA are believed to play an important role in the aging process.
Also mutations occur with every cell division so no two cells in an individual are identical, nor are clones. Thus, nuclear transfer clones from different maternal lineages are not clones in the strictest sense because the mitochondrial genome is not the same as that of the nucleus donor cell from which it was produced. This may have important implications for cross-species nuclear transfer in which nuclear-mitochondrial incompatibilities may lead to inviability.
Species cloned
The modern cloning techniques involving nuclear transfer have been successfully performed on several species. Landmark experiments in chronological order:
Tadpole: (1952) Many scientists questioned whether cloning had actually occurred and unpublished experiments by other labs were not able to reproduce the reported results.
Carp: (1963) In China, embryologist Tong Dizhou cloned a fish. He published the findings in an obscure Chinese science journal which was never translated into English.
Mice: (1986) was the first successfully cloned mammal; Soviet scientists Chaylakhyan, Veprencev, Sviridova, Nikitin had mice "Masha" cloned. Research was published in the magazine "Biofizika" volume ХХХII, issue 5 of 1987.
Sheep: (1996) From early embryonic cells by Steen Willadsen. Megan and Morag cloned from differentiated embryonic cells in June 1995 and Dolly the sheep in 1997.
Rhesus Monkey: Tetra (female, January 2000) from embryo splitting
Cattle: Alpha and Beta (males, 2001) and (2005) Brazil
Cat: CopyCat "CC" (female, late 2001), Little Nicky, 2004, was the first cat cloned for commercial reasons
Mule: Idaho Gem, a john mule born 2003-05-04, was the first horse-family clone.
Horse: Prometea, a Halflinger female born 2003-05-28, was the first horse clone.
For a complete list see: List of animals that have been cloned. During the first several divisions of a fertilized egg, no differentiation occurs and the cells can be separated without harm, but each will grow into an identical individual. This process has been used on cattle for decades to produce hundreds of identical individuals in some cases. This process is not considered cloning, but is called budding. The new individual is not derived from a differentiated cell, but from an undifferentiated egg. There is no way to determine which are the clones and which is the original.
Health aspects
The success rate of cloning has been low: Dolly the sheep was born after 277 eggs were used to create 29 embryos, which only produced three lambs at birth, only one of which lived, Dolly. Seventy calves have been created from 9,000 attempts and one third of them died young; Prometea took 328 attempts, and, more recently, Paris Texas was created after 400 attempts. Notably, although the first clones were frogs, no adult cloned frog has yet been produced from a somatic adult nucleus donor cell.
There were early claims that Dolly the Sheep had accelerated aging. Aging of this type is thought to be due to shortening of telomeres, regions at the tips of chromosomes which prevent genetic threads fraying every time a cell divides. Over time telomeres get worn down until cell-division is no longer possible — this is thought to be a cause of aging. However, subsequent studies showed that, if anything, Dolly's telomere were longer than normal. Dolly died in the year of 2003. Ian Wilmut said that Dolly's early death had nothing to do with cloning but with a respiratory infection common to lambs raised indoors like Dolly.
Consistent with Dolly's telomeres being longer, analysis of the telomeres from cloned cows showed that they were also longer. This suggests clones could live longer life spans although many died young after excessive growth. Researchers think that this could eventually be developed to reverse aging in humans, provided that this is based chiefly on the shortening of telomeres. Although some work has been performed on telomeres and aging in nuclear transfer clones, the evidence is at an early stage.
Dolly the Sheep
Dolly (1996-07-05 – 2003-02-14), an ewe, was the first mammal to have been successfully cloned from an adult cell (while the mice in USSR was cloned from embryo cell back in 1986 ). She was cloned at the Roslin Institute in Scotland and lived there until her death when she was 6. Her birth was announced on 1997-02-22.
The name "Dolly" came from a suggestion by Jesse Haase who helped with her birth, in honor of Dolly Parton, because it was a mammary cell that was cloned. The technique that was made famous by her birth is somatic cell nuclear transfer, in which a non-reproductive cell containing a nucleus is placed in a de-nucleated ovum (which then develops into a fetus). When Dolly was cloned in 1996 from a cell taken from a six-year-old ewe, she became the center of much controversy that still exists today.
Dolly's success is truly remarkable because it proved that the genetic material from a specialized adult cell, such as an udder cell programmed to express only those genes needed by udder cells, could be reprogrammed to generate an entire new organism. Before this demonstration, scientists believed that once a cell became specialized as a liver, heart, udder, bone, or any other type of cell, the change was permanent and other unneeded genes in the cell would become inactive. Some scientists believe that errors or incompleteness in the reprogramming process cause the high rates of death, deformity, and disability observed among animal clones.
On 2003-04-09 her stuffed remains were placed at Edinburgh's Royal Museum, part of the National Museums of Scotland.
Ian Wilmut's role in cloning Dolly the sheep is in doubt. In 2006 he admitted under oath in a Scottish court that he did not clone Dolly the Sheep and was not responsible for the scientific breakthrough which made it all possible. He credited Keith Campbell as being the brains behind Dolly the Sheep.
In August 2006, Iranian scientists oversaw the birth of the Middle East’s first cloned animal – a lamb that died minutes after it was born. However, in September 2006, Iranian scientists successfully cloned a sheep, by somatic cell nuclear transfer, at the Royan research institute in Isfahan, Iran’s second cloned lamb is still alive.
Human cloning
Human cloning is the creation of a genetically identical copy of an existing, or previously existing human, by growing cloned tissue from that individual. The term is generally used to refer to artificial human cloning; human clones in the form of identical twins are commonplace, with their cloning occurring during the natural process of reproduction.
Human cloning is amongst the most controversial forms of the practice. There have been numerous demands for all progress in the human cloning fields to be halted. One of the most ethically questionable problems with human cloning is farming of organs from clones. For example, many believe it is unethical to use a human clone to save the life of another. In this scenario, the cloned human would be euthanized so that the vital organs could be harvested. This process of renewing the body's organs would potentially increase the life expectancy of a human by 50 years.
The cloning described above is reproductive cloning, not to be confused with research cloning in which only parts (such as an organ) are cloned using genetic material from a patient's tissues.
Ethical issues of human cloning
Roman Catholicism and many conservative Christian groups have opposed human cloning and the cloning of human embryos, believing that a human life begins the moment a human egg becomes fertilized. Other Christian denominations such as the United Church of Christ do not believe a fertilized egg constitutes a living being, but still they oppose the cloning of embryonic cells. The World Council of Churches, representing nearly 400 denominations worldwide, opposed cloning of both human embryos and whole humans in February 2006. The United Methodist Church opposed research and reproductive cloning in May 2000 and again in May 2004.
Libertarian views on the subject suggest that the federal government of the United States does not have the power to regulate cloning, as it is not given any such authority by the US constitution. (Similar to abortion rights.)
At present, the main objection to human cloning is that the cloned individual may be biologically damaged, due to the inherent unreliability of its origin: researchers currently are unable to safely and reliably clone non-human primates.
However, many believe that as cloning research and methods improve, concerns of safety and reliability will no longer be an issue. However, it must be pointed out that this has yet to occur, and may never occur. Rudolph Jaenisch, a professor at Harvard, has pointed out that we have become more efficient at producing clones which are still defective (Development Dynamics. Volume 235, pages 2460-2469. 2006). Other arguments against cloning come from various religious orders (believing cloning violates God's will or the natural order of life), and a general discomfort some have with the idea of "meddling" with the creation and basic function of life. This unease often manifests itself in contemporary novels, movies, and popular culture, much like numerous other scientific discoveries and inventions before. Various fictional scenarios portray clones being unhappy, soulless, or unable to integrate into society. Furthermore, clones are often depicted not as unique individuals but as "spare parts," providing organs for the clone's original (or any non-clone that requires replacement organs).
Needless to say, cloning is a poignant and important topic, reflected by its frequent discussion and debate among politicians, scientists, the media, religions, and the general public.
Cloning extinct and endangered species
Cloning, or more precisely, the reconstruction of functional DNA from extinct species has, for decades, been a dream of some scientists. The possible implications of this were dramatized in the best-selling novel by Michael Crichton and high budget Hollywood thriller Jurassic Park. In real life, one of the most anticipated targets for cloning was once the Woolly mammoth, but attempts to extract DNA from frozen mammoths have been unsuccessful, though a joint Russo-Japanese team is currently working toward this goal.
In 2000, a cow named dendi Bessie gave birth to a cloned Asian gaur, an endangered species, but the calf died after two days. In 2003, a banteng was successfully cloned, followed by three African wildcats from a thawed frozen embryo. These successes provided hope that similar techniques (using surrogate mothers of another species) might be used to clone extinct species. Anticipating this possibility, tissue samples from the last bucardo (Pyrenean Ibex) were frozen immediately after it died. Researchers are also considering cloning endangered species such as the giant panda, ocelot, and cheetah. The "Froz
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