Cloning

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The sea anemone (Anthopleura elegantissima) undergoing cloning

In biology, cloning is the process of producing similar populations of genetically identical individuals that occurs in nature when organisms such as bacteria, insects or plants reproduce asexually. Cloning in biotechnology refers to processes used to create copies of DNA fragments (molecular cloning), cells (cell cloning), or organisms. The term also refers to the production of multiple copies of a product such as digital media or software.

The term clone is derived from the Ancient Greek word κλών klōn, "twig", referring to the process whereby a new plant can be created from a 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".[1][2] Since the term entered the popular lexicon in a more general context, the spelling clone has been used exclusively.

In botany, the term lusus was traditionally used.[3]:21, 43

In the United States, the human consumption of meat and other products from cloned animals was approved by the FDA on December 28, 2006, with no special labeling required because food from cloned organisms has been found to be identical to the organisms from which they were cloned.[4] Such practice has met strong resistance in other regions due to misinformation,[5] such as Europe, particularly over the labeling issue.

Molecular cloning

Molecular cloning refers to the process of making multiple molecules. Cloning is commonly used to amplify DNA fragments containing whole genes, but it can also be used to amplify any DNA sequence such as promoters, non-coding sequences and randomly fragmented DNA. It is used in a wide array of biological experiments and practical applications ranging from genetic fingerprinting to large scale protein production. Occasionally, 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, such as in positional cloning. In practice, localization of the gene to a chromosome or genomic region does not necessarily enable one to isolate or amplify the relevant genomic sequence. To amplify any DNA sequence in a living organism, that sequence must be linked to an origin of replication, which is a sequence of DNA capable of directing the propagation of itself and any linked sequence. However, a number of other features are needed and a variety of specialised cloning vectors (small piece of DNA into which a foreign DNA fragment can be inserted) exist that allow protein expression, tagging, single stranded RNA and DNA production and a host of other manipulations.

Cloning of any DNA fragment essentially involves four steps[6]

  1. fragmentation - breaking apart a strand of DNA
  2. ligation - gluing together pieces of DNA in a desired sequence
  3. transfection - inserting the newly formed pieces of DNA into cells
  4. screening/selection - selecting out the cells that were successfully transfected with the new DNA

Although these steps are invariable among cloning procedures a number of alternative routes can be selected; these are summarized as a cloning strategy.

Initially, the DNA of interest needs to be isolated to provide a DNA segment of suitable size. Subsequently, a ligation procedure is used where the amplified fragment is inserted into a vector (piece of DNA). The vector (which is frequently circular) is linearised using restriction enzymes, and incubated with the fragment of interest under appropriate conditions with an enzyme called DNA ligase. Following ligation the vector with the insert of interest is transfected into cells. A number of alternative techniques are available, such as chemical sensitivation of cells, electroporation, optical injection and biolistics. Finally, the transfected cells are cultured. As the aforementioned procedures are of particularly low efficiency, there is a need to identify the cells that have been successfully transfected with the vector construct containing the desired insertion sequence in the required orientation. Modern cloning vectors include selectable antibiotic resistance markers, which allow only cells in which the vector has been transfected, to grow. Additionally, the cloning vectors may contain colour selection markers, which provide blue/white screening (alpha-factor complementation) on X-gal medium. Nevertheless, these selection steps do not absolutely guarantee that the DNA insert is present in the cells obtained. Further investigation of the resulting colonies must be required to confirm that cloning was successful. This may be accomplished by means of PCR, restriction fragment analysis and/or DNA sequencing.

Cell cloning

Cloning unicellular organisms

Cloning cell-line colonies using cloning rings

Cloning a cell means to derive a population of cells from a single cell. In the case of unicellular organisms such as bacteria and yeast, this process is remarkably simple and essentially only requires the inoculation of the appropriate medium. However, in the case of cell cultures from multi-cellular organisms, cell cloning is an arduous task as these cells will not readily grow in standard media.

A useful tissue culture technique used to clone distinct lineages of cell lines involves the use of cloning rings (cylinders).[4] According to this technique, a single-cell suspension of cells that have been exposed to a mutagenic agent or drug used to drive selection is plated at high dilution to create isolated colonies; each arising from a single and potentially clonal distinct cell. At an early growth stage when colonies consist of only a few of cells, sterile polystyrene rings (cloning rings), which have been dipped in grease are placed over an individual colony and a small amount of trypsin is added. Cloned cells are collected from inside the ring and transferred to a new vessel for further growth.

Cloning stem cells

Somatic-cell nuclear transfer, known as SCNT, can also be used to create embryos for research or therapeutic purposes. The most likely purpose for this is to produce embryos for use in stem cell research. This process is also called "research cloning" or "therapeutic cloning." The goal is not to create cloned human beings (called "reproductive cloning"), but rather to harvest stem cells that can be used to study human development and to potentially treat disease. While a clonal human blastocyst has been created, stem cell lines are yet to be isolated from a clonal source.[7]

Therapeutic cloning is achieved by creating embryonic stem cells in the hopes of treating diseases such as diabetes and Alzheimer’s. The process begins by taking out the nucleus (containing the DNA) from an egg cell and putting in it a nucleus from the adult cell to be cloned.[8] In the case of someone with Alzheimer’s disease, the nucleus from a skin cell of that patient is placed into an empty egg. The reprogrammed cell begins to develop into an embryo because the egg reacts with the transferred nucleus. The embryo will become genetically identical to the patient.[8] The embryo will then form a blastocyst which has the potential to form/become any cell in the body.[9]

The reason why SCNT is used for cloning is because somatic cells can be easily acquired and cultured in the lab. This process can either add or delete specific genomes of farm animals. A key point to remember is that cloning is achieved when the oocyte maintains its normal functions and instead of using sperm and egg genomes to replicate, the oocyte is inserted into the donor’s somatic cell nucleus.[10] The oocyte will react on the somatic cell nucleus, the same way it would on sperm cells.[10]

The process of cloning a particular farm animal using SCNT is relatively the same for all animals. The first step is to collect the somatic cells from the animal that will be cloned. The somatic cells could be used immediately or stored in the laboratory for later use.[10] The hardest part of SCNT is removing maternal DNA from an oocyte at metaphase II. Once this has been done, the somatic nucleus can be inserted into an egg cytoplasm.[10] This creates a one-cell embryo. The grouped somatic cell and egg cytoplasm are then introduced to an electrical current.[10] This energy will hopefully allow the cloned embryo to begin development. The successfully developed embryos are then placed in surrogate recipients, such as a cow or sheep in the case of farm animals.[10]

SCNT is seen as a good method for producing agriculture animals for food consumption. It successfully cloned sheep, cattle, goats, and pigs. Another benefit is SCNT is seen as a solution to clone endangered species that are on the verge of going extinct.[10] However, stresses placed on both the egg cell and the introduced nucleus are enormous, leading to a high loss in resulting cells. For example, the cloned sheep Dolly was born after 277 eggs were used for SCNT, which created 29 viable embryos. Only three of these embryos survived until birth, and only one survived to adulthood.[11] As the procedure currently cannot be automated, and has to be performed manually under a microscope, SCNT is very resource intensive. The biochemistry involved in reprogramming the differentiated somatic cell nucleus and activating the recipient egg is also far from being well-understood.

In SCNT, not all of the donor cell's genetic information is transferred, as the donor cell's mitochondria that contain their own mitochondrial DNA are left behind. The resulting hybrid cells retain those mitochondrial structures which originally belonged to the egg. As a consequence, clones such as Dolly that are born from SCNT are not perfect copies of the donor of the nucleus.

Organism cloning

Organism cloning (also called reproductive cloning) refers to the procedure of creating a new multicellular organism, genetically identical to another. In essence this form of cloning is an asexual method of reproduction, where fertilization or inter-gamete contact does not take place. Asexual reproduction is a naturally occurring phenomenon in many species, including most plants (see vegetative reproduction) and some insects. Scientists have made some major achievements with cloning, including the asexual reproduction of sheep and cows. There is a lot of ethical debate over whether or not cloning should be used. However, cloning, or asexual propagation,[12] has been common practice in the horticultural world for hundreds of years.

Horticultural

The term clone is used in horticulture to refer to descendants of a single plant which were 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.[13] As an example, some European cultivars of grapes represent clones that have been propagated for over two millennia. Other examples are potato and banana.[14] Grafting can be regarded as cloning, since all the shoots and branches coming from the graft are genetically a clone of a single individual, 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 naturally. Parts of an individual plant may become detached by fragmentation and grow on to become separate clonal individuals. A common example is in the vegetative reproduction of moss and liverwort gametophyte clones by means of gemmae. Some vascular plants e.g. dandelion and certain viviparous grasses also form seeds asexually, termed apomixis, resulting in clonal populations of genetically identical individuals.

Parthenogenesis

Clonal derivation exists in nature in some animal species and is referred to as parthenogenesis (reproduction of an organism by itself without a mate). This is an asexual form of reproduction that is only found in females of some insects, crustaceans, nematodes,[15] fish (for example the Hammerhead shark[16]), the Komodo dragon[16] and lizards. The growth and development occurs without fertilization by a male. In plants, parthenogenesis means the development of an embryo from an unfertilized egg cell, and is a component process of apomixis. In species that use the XY sex-determination system, the offspring will always be female. An example is the "Little Fire Ant" (Wasmannia auropunctata), which is native to Central and South America but has spread throughout many tropical environments.

Artificial cloning of organisms

Artificial cloning of organisms may also be called reproductive cloning.

First moves

Hans Spemann, a German embryologist was awarded a Nobel Prize in Physiology or Medicine in 1935 for his discovery of the effect now known as embryonic induction, exercised by various parts of the embryo, that directs the development of groups of cells into particular tissues and organs. In 1928 he and his student, Hilde Mangold, were the first to perform somatic-cell nuclear transfer using amphibian embryos – one of the first moves towards cloning.[17]

Methods

Reproductive cloning generally uses "somatic cell nuclear transfer" (SCNT) to create animals that are genetically identical. This process entails the transfer of a nucleus from a donor adult cell (somatic cell) to an egg from which the nucleus has been removed, or to a cell from a blastocyst from which the nucleus has been removed.[18] If the egg begins to divide normally it is transferred into the uterus of the surrogate mother. Such clones are not strictly identical since the somatic cells may contain mutations in their nuclear DNA. Additionally, the mitochondria in the cytoplasm also contains DNA and during SCNT this mitochondrial DNA is wholly from the cytoplasmic donor's egg, thus 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 death.

Artificial embryo splitting or embryo twinning, a technique that creates monozygotic twins from a single embryo, is not considered in the same fashion as other methods of cloning. During that procedure, an donor embryo is split in two distinct embryos, that can then be transferred via embryo transfer. It is optimally performed at the 6- to 8-cell stage, where it can be used as an expansion of IVF to increase the number of available embryos.[19] If both embryos are successful, it gives rise to monozygotic (identical) twins.

Dolly the sheep

Dolly clone

Dolly, a Finn-Dorset ewe, was the first mammal to have been successfully cloned from an adult cell. Dolly was formed by taking a cell from the udder of her biological mother. Her biological mother was 6 years old when the cells were taken from her udder.[20] Dolly's embryo was created by taking the cell and inserting it into a sheep ovum. It took 434 attempts before an embryo was successful.[21] The embryo was then placed inside a female sheep that went through a normal pregnancy.[22] She was cloned at the Roslin Institute in Scotland and lived there from her birth in 1996 until her death in 2003 when she was six. She was born on July 5, 1996 but not announced to the world until February 22, 1997.[21] Her stuffed remains were placed at Edinburgh's Royal Museum, part of the National Museums of Scotland.[23]

Dolly was publicly significant because the effort showed that genetic material from a specific adult cell, programmed to express only a distinct subset of its genes, can be reprogrammed to grow an entirely new organism. Before this demonstration, it had been shown by John Gurdon that nuclei from differentiated cells could give rise to an entire organism after transplantation into an enucleated egg.[24] However, this concept was not yet demonstrated in a mammalian system.

Cloning Dolly the sheep had a low success rate per fertilized egg; she was born after 277 eggs were used to create 29 embryos, which only produced three lambs at birth, only one of which lived. Seventy calves have been created and one third of them died young; Prometea took 814 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 pathologies resembling accelerated aging. Scientists speculated that Dolly's death in 2003 was related to the shortening of telomeres, DNA-protein complexes that protect the end of linear chromosomes. However, other researchers, including Ian Wilmut who led the team that successfully cloned Dolly, argue that Dolly's early death due to respiratory infection was unrelated to deficiencies with the cloning process.

Dolly was named after Dolly Parton because the cells cloned to make her were from an udder.[25]

Species cloned

The modern cloning techniques involving nuclear transfer have been successfully performed on several species. Notable experiments include:

Human cloning

Human cloning is the creation of a genetically identical copy of an existing or previously existing human, human cell, or human tissue. The term is generally used to refer to artificial human cloning. Note that human clones in the form of identical twins are commonplace, with their cloning occurring during the natural process of reproduction — i.e. identical twins are not discussed as artificial clones. There are two commonly discussed types of human cloning: therapeutic cloning and reproductive cloning. Therapeutic cloning involves cloning adult cells for use in medicine, and is an active area of research. Reproductive cloning would involve making cloned humans.[49]

The first hybrid human clone was created in November 1998, by Advanced Cell Technology. It was created using somatic cell nuclear transfer (SCNT). A nucleus was taken from a man's leg cell, and inserted into a cow's enucleated egg (i.e. whose DNA was removed). The hybrid cell was cultured and developed into an embryo. The embryo was destroyed after 12 days.[50]

On January, 2008, Dr. Samuel Wood and Andrew French of the biotechnology company Stemagen, announced that they successfully created the first 5 mature human embryos using DNA from adult skin cells, aiming to provide a source of viable embryonic stem cells, using SCNT. In this case, each embryo was created by taking a nucleus from a skin cell (donated by. Wood and a colleague) and inserted it into a human egg from which the nucleus had been removed. It is not clear if the embryos produced would have been capable of further development.[51]

In May 2013, a group of scientists led by Shoukhrat Mitalipov published a report of successful creation of embryos using SCNT.[52] All the DNA used came from fetuses or infants.[53] In April 2014, a team led by Robert Lanza, CEO of Advanced Cell Technology, reported that they had replicated Mitalipov's results and further demonstrated the effectiveness by cloning adult cells.[53][54]

Somatic cell nuclear transfer (SCNT) is the implantation of the nucleus of a somatic cell into a host, enucleated egg cell. The cell is then stimulated via electric current to fuse the host and donor material. This process has been widely studied and been proven to be ineffective compared to the technique of induced pluripotent stem cells (iPSCs).[55] iPSCs are created by reprogramming human, adult somatic cells into stem cells. Development of this process could lead to more effective applications of personalized medicine.[56]

Ethical issues of cloning

In bioethics, the ethics of cloning' refers to a variety of ethical positions regarding the practice and possibilities of cloning, especially human cloning. While many of these views are religious in origin, the questions raised by cloning are faced by secular perspectives as well. Perspectives on human cloning are theoretical, as human therapeutic and reproductive cloning are not commercially used; animals are currently cloned in laboratories and in livestock production.

Advocates support development of therapeutic cloning in order to generate tissues and whole organs to treat patients who otherwise cannot obtain transplants,[57] to avoid the need for immunosuppressive drugs,[49] and to stave off the effects of aging.[58] Advocates for reproductive cloning believe that parents who cannot otherwise procreate should have access to the technology.[59]

Opponents of cloning have concerns that technology is not yet developed enough to be safe,[60] that it could be prone to abuse (leading to the generation of humans from whom organs and tissues would be harvested),[61][62] and have concerns about how cloned individuals could integrate with families and with society at large.[63][64]

Religious groups are divided, with some opposing the technology as usurping God's place and, to the extent embryos are used, destroying a human life; others support therapeutic cloning's potential life-saving benefits.[65][66]

Cloning of animals is opposed by animal-groups due to the number of cloned animals that suffer from malformations before they die,[67][68] and while food from cloned animals has been approved by the US FDA,[5][69] its use is opposed by groups concerned about food safety.[70][71][72]

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. Current cloning techniques have an average success rate of less than 5 percent, even when working with familiar species; cloning wild animals is usually less than 1 percent successful.[73] The possible implications of this were dramatized in the best-selling novel by Michael Crichton and high budget Hollywood thriller Jurassic Park. Early hopes to save species through cloning have been dashed as the difficulties of cloning any species have made progress very slow.[74][75] Several tissue banks have come into existence, including the "Frozen Zoo" at the San Diego Zoo, to store frozen tissue from the world's rarest and most endangered species.[74][76][77]

In 2001, a cow named 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 in liquid nitrogen immediately after it died in 2000. Researchers are also considering cloning endangered species such as the giant panda and cheetah.

In 2002, geneticists at the Australian Museum announced that they had replicated DNA of the Thylacine (Tasmanian Tiger), at the time extinct for about 65 years, using polymerase chain reaction.[78] However, on February 15, 2005 the museum announced that it was stopping the project after tests showed the specimens' DNA had been too badly degraded by the (ethanol) preservative. On 15 May 2005 it was announced that the Thylacine project would be revived, with new participation from researchers in New South Wales and Victoria.

In January 2009, for the first time, an extinct animal, the Pyrenean ibex mentioned above was cloned, at the Centre of Food Technology and Research of Aragon, using the preserved DNA of the skin samples from 2001 and domestic goat egg-cells. The ibex died shortly after birth due to physical defects in its lungs.[79]

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 January 2011, it was reported by Yomiuri Shimbun that a team of scientists headed by Akira Iritani of Kyoto University had built upon research by Dr. Wakayama, saying that they will extract DNA from a mammoth carcass that had been preserved in a Russian laboratory and insert it into the egg cells of an African elephant in hopes of producing a mammoth embryo. The researchers said they hoped to produce a baby mammoth within six years.[80][81]

Scientists at the University of Newcastle and University of New South Wales announced in March 2013 that the very recently extinct gastric-brooding frog would be the subject of a cloning attempt to resurrect the species.[82]

Lifespan

After an eight-year project involving the use of a pioneering cloning technique, Japanese researchers created 25 generations of healthy cloned mice with normal lifespans, demonstrating that clones are not intrinsically shorter-lived than naturally born animals.[83]

In popular culture

In an article in the November 8, 1993 article of Time Magazine, cloning was portrayed in a negative way, modifying Michelangelo's Creation of Adam to depict Adam with five identical hands. Newsweek Magazine's March 10, 1997 issue also critiqued the ethics of human cloning, and included a graphic depicting identical babies in beakers.

Cloning is a recurring theme in a wide variety of contemporary science fiction, ranging from action films such as the 2000 film The 6th Day and Resident Evil (film series), Jurassic Park (film), and The Island (2005 film), to comedies such as Woody Allen's 1973 film Sleeper.[84]

Science fiction has used cloning, most commonly and specifically human cloning, due to the fact that it brings up controversial questions of identity.[85][86] In Aldous Huxley’s Brave New World (1932), human cloning is a major plot device that not only drives the story but also makes the reader think critically about what identity means; this concept was re-examined fifty years later in C. J. Cherryh’s novels Forty Thousand in Gehenna (1983) and Cyteen (1988). Kazuo Ishiguro's 2005 novel Never Let Me Go centres on human clones and considers the ethics of the practice. Another book that embodies the ideas of cloning is The House of the Scorpion which explores the rights of human clones and organ harvesting, set from the eyes of a clone. The short novel Containing God by Dr.S.M.Wasi Haider similarly deals with the ideas of cloning and the ethics, lust, and other issues revolving around the topic, emphasizing the idea that creating life gives people the false sense of divinity. The implications of using clones to replace deceased loved ones are explored in several works of fiction. In Margaret Peterson Haddix's novel Double Identity, the main character discovers that she is a clone of her deceased older sister. A Number is a 2002 play by English playwright Caryl Churchill which addresses the subject of human cloning and identity, especially nature and nurture. The story, set in the near future, is structured around the conflict between a father (Salter) and his sons (Bernard 1, Bernard 2, and Michael Black) – two of whom are clones of the first one. A Number was adapted by Caryl Churchill for television, in a co-production between the BBC and HBO Films.[87] Starring Rhys Ifans and Tom Wilkinson, it was broadcast on BBC Two on 10 Sep 2008.[88]

A recurring sub-theme of cloning fiction is the use of clones as a supply of organs for transplantation. The 2005 Kazuo Ishiguro novel Never Let Me Go and the 2010 film adaption[89] are set in an alternate history in which cloned humans are created for the sole purpose of providing organ donations to naturally born humans, despite the fact that they are fully sentient and self-aware. The 2005 film The Island[90] revolves around a similar plot, with the exception that the clones are unaware of the reason for their existence. In the futuristic novel The House of the Scorpion, clones are used to grow organs for their wealthy "owners", and the main character was a complete clone.

The use of human cloning for military purposes has also been explored in several works. Star Wars portrays human cloning in Clone Wars,[91] Star Wars Episode II: Attack of the Clones and Star Wars Episode III: Revenge of the Sith, in the form of the Grand Army of the Republic, an army of cloned soldiers. The Expanded Universe also has numerous examples of cloning, including the Thrawn trilogy, The Hand of Thrawn duology, and Clone Wars-era media.

The exploitation of human clones for dangerous and undesirable work was examined in the 2009 British science fiction film Moon.[92] In the futuristic novel Cloud Atlas and subsequent film, one of the story lines focuses on a genetically-engineered Fabricant Clone named Sonmi~451 who is one of millions raised in an artificial "wombtank," destined to serve from birth. She is one of thousands of clones created for manual and emotional labor; Sonmi herself works as a server in a restaurant. She later discovers that the sole source of food for clones, called 'Soap', is manufactured from the clones themselves.[93]

In the comedy film Multiplicity, a man clones himself three times with the help of a geneticist.

Cloning has been used in fiction as a way of recreating historical figures. In the 1976 Ira Levin novel The Boys from Brazil and its 1978 film adaptation,[94] Josef Mengele uses cloning to create copies of Adolf Hitler. A Parade of Mirrors and Reflections, a novel by Anatoly Kudryavitsky, centers on the cloning of deceased Soviet premier Yuri Andropov.

In the anime A Certain Scientific Railgun, Level 5 esper Mikoto Misaka was cloned commercially over 20,000 times for the purposes of research into the possibility of a "Level 6" esper. In another anime/manga series Neon Genesis Evangelion, human cloning is a topic which features heavily around the origin of the character Ayanami Rei. In 2012, a Japanese television show named "Bunshin" was created. The story's main character, Mariko, is a woman studying child welfare in Hokkaido. She grew up always doubtful about the love from her mother, who looked nothing like her and who died nine years before. One day, she finds some of her mother's belongings at a relative's house, and heads to Tokyo to seek out the truth behind her birth. She later discovered that she was a clone.[95]

Cloning is also featured in the Halo franchise, particularly a technique known as "flash cloning" in which the unstable clone of an individual is created in an incredibly short span of time. Flash cloning is used by the UNSC to kidnap young children for induction into the SPARTAN-II military program, who are surreptitiously replaced by flash clones which die within a short span of time to ensure that no one looks for the children. The MMORPG EVE Online and online FPS DUST 514 takes place in the distant future where the player characters are all clones; at the moment of death, the person's brain-state is mapped, transmitted and applied to a 'blank' clone in a station or facility some distance away.

In the 2013 television show Orphan Black,[96] cloning is used as a scientific double blind study on the behavioral adaptation of the clones.

See also

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