Cloning

Source: Wikipedia, the free encyclopedia.
(Redirected from
Conservation cloning
)

quaking aspen
.

Cloning is the process of producing individual organisms with identical or virtually identical DNA, either by natural or artificial means. In nature, some organisms produce clones through asexual reproduction. In the field of biotechnology, cloning is the process of creating cloned organisms (copies) of cells and of DNA fragments (molecular cloning).

Etymology

Coined by

Herbert J. Webber, the term clone derives from the Ancient Greek word κλών (klōn), twig, which is the process whereby a new plant is created from a twig. In botany, the term lusus was used.[1] In horticulture, the spelling clon was used until the early twentieth century; the final e came into use to indicate the vowel is a "long o" instead of a "short o".[2][3]
Since the term entered the popular lexicon in a more general context, the spelling clone has been used exclusively.

Natural cloning

Cloning is a natural form of reproduction that has allowed life forms to spread for hundreds of millions of years. It is a reproduction method used by plants, fungi, and bacteria, and is also the way that clonal colonies reproduce themselves.[4][5] Examples of these organisms include blueberry plants, Hazel trees, the Pando trees,[6][7] the Kentucky coffeetree, Myrica, and the American sweetgum.

Molecular cloning

Molecular cloning refers to the process of making multiple molecules. Cloning is commonly used to amplify

affinity tagging, single-stranded RNA
or DNA production and a host of other molecular biology tools.

Cloning of any DNA fragment essentially involves four steps[8]

  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

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 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).

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 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

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.[10]

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 removing the nucleus (containing the DNA) from an egg cell and inserting a nucleus from the adult cell to be cloned.[11] 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.[11] The embryo will then form a blastocyst which has the potential to form/become any cell in the body.[12]

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 donor's somatic cell nucleus is inserted into the oocyte.[13] The oocyte will react to the somatic cell nucleus, the same way it would to a sperm cell's nucleus.[13]

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.[13] 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.[13] This creates a one-cell embryo. The grouped somatic cell and egg cytoplasm are then introduced to an electrical current.[13] 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.[13]

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.[13] However, stresses placed on both the egg cell and the introduced nucleus can be enormous, which led to a high loss in resulting cells in early research. 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.[14] As the procedure could not be automated, and had to be performed manually under a microscope, SCNT was very resource intensive. The biochemistry involved in reprogramming the differentiated somatic cell nucleus and activating the recipient egg was also far from being well understood. However, by 2014 researchers were reporting cloning success rates of seven to eight out of ten[15] and in 2016, a Korean Company Sooam Biotech was reported to be producing 500 cloned embryos per day.[16]

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 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,[17] has been common practice in the horticultural world for hundreds of years.

Horticultural

cuttings
, such as grape vines, is an ancient form of cloning

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.[18] As an example, some European cultivars of grapes represent clones that have been propagated for over two millennia. Other examples are potato and banana.[19]

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,

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

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 steps

somatic-cell nuclear transfer using amphibian embryos – one of the first steps towards cloning.[22]

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.[23] 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, a donor

monozygotic (identical) twins
.

Dolly the sheep

Dolly was publicly significant because the effort showed that genetic material from a specific adult cell, designed to express only a distinct subset of its genes, can be redesigned 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.[30] However, this concept was not yet demonstrated in a mammalian system.

The first mammalian cloning (resulting in Dolly) had a success rate of 29 embryos per 277 fertilized eggs, which produced three lambs at birth, one of which lived. In a bovine experiment involving 70 cloned calves, one-third of the calves died quite young. The first successfully cloned horse, Prometea, took 814 attempts. Notably, although the first[clarification needed] clones were frogs, no adult cloned frog has yet been produced from a somatic adult nucleus donor cell.

There were early claims that Dolly 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 problems with the cloning process. This idea that the nuclei have not irreversibly aged was shown in 2013 to be true for mice.[31]

Dolly was named after performer Dolly Parton because the cells cloned to make her were from a mammary gland cell, and Parton is known for her ample cleavage.[32]

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 a human. The term is generally used to refer to artificial human cloning, which is the reproduction of human cells and tissues. It does not refer to the natural conception and delivery of

identical twins. The possibility of human cloning has raised controversies. These ethical concerns have prompted several nations to pass legislation regarding human cloning and its legality. As of right now, scientists have no intention of trying to clone people and they believe their results should spark a wider discussion about the laws and regulations the world needs to regulate cloning.[68]

Two commonly discussed types of theoretical human cloning are therapeutic cloning and reproductive cloning. Therapeutic cloning would involve cloning cells from a human for use in medicine and transplants, and is an active area of research, but is not in medical practice anywhere in the world, as of 2021[update]. Two common methods of therapeutic cloning that are being researched are

somatic-cell nuclear transfer and, more recently, pluripotent stem cell induction. Reproductive cloning would involve making an entire cloned human, instead of just specific cells or tissues.[69]

Ethical issues of cloning

There are a variety of ethical positions regarding the 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 to generate tissues and whole organs to treat patients who otherwise cannot obtain transplants,

immunosuppressive drugs,[69] and to stave off the effects of aging.[71] Advocates for reproductive cloning believe that parents who cannot otherwise procreate should have access to the technology.[72]

Opponents of cloning have concerns that technology is not yet developed enough to be safe[73] and that it could be prone to abuse (leading to the generation of humans from whom organs and tissues would be harvested),[74][75] as well as concerns about how cloned individuals could integrate with families and with society at large.[76][77]

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.[78][79]

Cloning of animals is opposed by animal-groups due to the number of cloned animals that suffer from malformations before they die, and while food from cloned animals has been approved by the US FDA,[80][81] its use is opposed by groups concerned about food safety.[82][83]

Cloning extinct and endangered species

Cloning, or more precisely, the reconstruction of functional DNA from

extinct species has, for decades, been a dream. Possible implications of this were dramatized in the 1984 novel Carnosaur and the 1990 novel Jurassic Park.[84][85] The best current cloning techniques have an average success rate of 9.4 percent[86] (and as high as 25 percent[31]) when working with familiar species such as mice,[note 1] while cloning wild animals is usually less than 1 percent successful.[89]

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.[84][90][91] This is also referred to as "Conservation cloning".[92][93]

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.[94][95][96][97]

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.[98] However, on 15 February 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.[99]

In 2003, 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 frozen cell nucleus of the skin samples from 2001 and domestic goat egg-cells. The ibex died shortly after birth due to physical defects in its lungs.[100]

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.[when?] 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 Asian elephant in hopes of producing a mammoth embryo. The researchers said they hoped to produce a baby mammoth within six years.[101][102] It was noted, however that the result, if possible, would be an elephant-mammoth hybrid rather than a true mammoth.[103] Another problem is the survival of the reconstructed mammoth: ruminants rely on a symbiosis with specific microbiota in their stomachs for digestion.[103]

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.[104]

Many such "De-extinction" projects are described in the Long Now Foundation's Revive and Restore Project.[105]

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.[31][106] Other sources have noted that the offspring of clones tend to be healthier than the original clones and indistinguishable from animals produced naturally.[107]

Some posited that Dolly the sheep may have aged more quickly than naturally born animals, as she died relatively early for a sheep at the age of six. Ultimately, her death was attributed to a respiratory illness, and the "advanced aging" theory is disputed.[108][dubious ]

A detailed study released in 2016 and less detailed studies by others suggest that once cloned animals get past the first month or two of life they are generally healthy. However, early pregnancy loss and neonatal losses are still greater with cloning than natural conception or assisted reproduction (IVF). Current research is attempting to overcome these problems.[32]

In popular culture

Discussion of cloning in the popular media often presents the subject negatively. In an article in the 8 November 1993 article of

Creation of Adam to depict Adam with five identical hands.[109] Newsweek's 10 March 1997 issue also critiqued the ethics of human cloning, and included a graphic depicting identical babies in beakers.[110]

The concept of cloning, particularly human cloning, has featured a wide variety of science fiction works. An early fictional depiction of cloning is

eggs in vitro, causing them to split into identical genetic copies of the original.[111][112] Following renewed interest in cloning in the 1950s, the subject was explored further in works such as Poul Anderson's 1953 story UN-Man, which describes a technology called "exogenesis", and Gordon Rattray Taylor's book The Biological Time Bomb, which popularised the term "cloning" in 1963.[113]

Cloning is a recurring theme in a number of contemporary science fiction films, ranging from action films such as Anna to the Infinite Power, The Boys from Brazil, Jurassic Park (1993), Alien Resurrection (1997), The 6th Day (2000), Resident Evil (2002), Star Wars: Episode II – Attack of the Clones (2002), The Island (2005) and Moon (2009) to comedies such as Woody Allen's 1973 film Sleeper.[114]

The process of cloning is represented variously in fiction. Many works depict the artificial creation of humans by a method of growing cells from a tissue or DNA sample; the replication may be instantaneous, or take place through slow growth of human embryos in artificial wombs. In the long-running British television series Doctor Who, the Fourth Doctor and his companion Leela were cloned in a matter of seconds from DNA samples ("The Invisible Enemy", 1977) and then – in an apparent homage to the 1966 film Fantastic Voyage – shrunk to microscopic size to enter the Doctor's body to combat an alien virus. The clones in this story are short-lived, and can only survive a matter of minutes before they expire.[115] Science fiction films such as The Matrix and Star Wars: Episode II – Attack of the Clones have featured scenes of human foetuses being cultured on an industrial scale in mechanical tanks.[116]

Cloning humans from body parts is also a common theme in science fiction. Cloning features strongly among the science fiction conventions parodied in Woody Allen's Sleeper, the plot of which centres around an attempt to clone an assassinated dictator from his disembodied nose.[117] In the 2008 Doctor Who story "Journey's End", a duplicate version of the Tenth Doctor spontaneously grows from his severed hand, which had been cut off in a sword fight during an earlier episode.[118]

Another example is the 2021 film Girl Next in which a woman is abducted, drugged and brainwashed into becoming an obedient, living sex doll. It is later proved that she is a clone of a clone designed to assassinate the traffickers.

After the death of her beloved 14-year-old Coton de Tulear named Samantha in late 2017, Barbra Streisand announced that she had cloned the dog, and was now "waiting for [the two cloned pups] to get older so [she] can see if they have [Samantha's] brown eyes and her seriousness".[119] The operation cost $50,000 through the pet cloning company ViaGen.[120]

Cloning and identity

Science fiction has used cloning, most commonly and specifically

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.[123]

In 2012, a Japanese television series 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.[124]

In the 2013 television series Orphan Black, cloning is used as a scientific study on the behavioral adaptation of the clones.[125] In a similar vein, the book The Double by Nobel Prize winner José Saramago explores the emotional experience of a man who discovers that he is a clone.[126]

Cloning as resurrection

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, Josef Mengele uses cloning to create copies of Adolf Hitler.[127]

In Michael Crichton's 1990 novel Jurassic Park, which spawned a series of Jurassic Park feature films, the bioengineering company InGen develops a technique to resurrect extinct species of dinosaurs by creating cloned creatures using DNA extracted from fossils. The cloned dinosaurs are used to populate the Jurassic Park wildlife park for the entertainment of visitors. The scheme goes disastrously wrong when the dinosaurs escape their enclosures. Despite being selectively cloned as females to prevent them from breeding, the dinosaurs develop the ability to reproduce through parthenogenesis.[128]

Cloning for warfare

The use of cloning for military purposes has also been explored in several fictional works. In Doctor Who, an alien race of armour-clad, warlike beings called Sontarans was introduced in the 1973 serial "The Time Warrior". Sontarans are depicted as squat, bald creatures who have been genetically engineered for combat. Their weak spot is a "probic vent", a small socket at the back of their neck which is associated with the cloning process.[129] The concept of cloned soldiers being bred for combat was revisited in "The Doctor's Daughter" (2008), when the Doctor's DNA is used to create a female warrior called Jenny.[130]

The 1977 film

space war waged by a massive army of heavily armoured clone troopers that leads to the foundation of the Galactic Empire. Cloned soldiers are "manufactured" on an industrial scale, genetically conditioned for obedience and combat effectiveness. It is also revealed that the popular character Boba Fett originated as a clone of Jango Fett, a mercenary who served as the genetic template for the clone troopers.[131][132]

Cloning for exploitation

A recurring sub-theme of cloning fiction is the use of clones as a supply of

transplantation. The 2005 Kazuo Ishiguro novel Never Let Me Go and the 2010 film adaption[133] 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[134]
revolves around a similar plot, with the exception that the clones are unaware of the reason for their existence.

The exploitation of human clones for dangerous and undesirable work was examined in the 2009 British science fiction film Moon.[135] In the futuristic novel Cloud Atlas and subsequent film, one of the story lines focuses on a genetically-engineered fabricant clone named Sonmi~451, one of millions raised in an artificial "wombtank", destined to serve from birth. She is one of thousands 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.[136]

In the film Us, at some point prior to the 1980s, the US Government creates clones of every citizen of the United States with the intention of using them to control their original counterparts, akin to voodoo dolls. This fails, as they were able to copy bodies, but unable to copy the souls of those they cloned. The project is abandoned and the clones are trapped exactly mirroring their above-ground counterparts' actions for generations. In the present day, the clones launch a surprise attack and manage to complete a mass-genocide of their unaware counterparts.[137][138]

In the series of A Certain Magical Index and A Certain Scientific Railgun, one of the espers, Mikoto Misaka, had DNA was harvested unknowingly, creating 20,000 exact but not equally powerful clones for an experiment. They were used as target practice by Accelerator, just to level up, as killing the original multiple times is impossible. The experiment ended when Toma Kamijo saved and foiled the experiment. The remaining clones have been dispersed everywhere in the world to conduct further experiments to expand their lifespans, save for at least 10 who remained in Academy City, and the last clone, who was not fully developed when the experiment stopped.[citation needed]

See also

Notes

  1. BGI, a Chinese company[87] and in another news article in 2015 a Korean Company, Sooam Biotech, claimed 40 percent success rates with cloning dogs[88]

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Further reading

  • Guo, Owen. "World's Biggest Animal Cloning Center Set for '16 in a Skeptical China". The New York Times, 26 November 2015
  • Lerner, K. Lee. "Animal cloning". The Gale Encyclopedia of Science, edited by K. Lee Lerner and Brenda Wilmoth Lerner, 5th ed., Gale, 2014. Science in Context, link[permanent dead link]
  • Dutchen, Stephanie (11 July 2018). "Rise of the Clones". Harvard Medical School.

External links