Cloning is the process of producing individual with identical , either by natural or artificial means. In nature, some organisms produce clones through asexual reproduction; this reproduction of an organism by itself without a mate is known as parthenogenesis. In the field of biotechnology, cloning is the process of creating cloned organisms of cells and of DNA fragments.
The artificial cloning of organisms, sometimes known as reproductive cloning, is often accomplished via somatic-cell nuclear transfer (SCNT), a cloning method in which a viable embryo is created from a somatic cell and an egg cell. In 1996, Dolly the sheep achieved notoriety for being the first mammal cloned from a somatic cell. Another example of artificial cloning is molecular cloning, a technique in molecular biology in which a single living cell is used to clone a large population of cells that contain identical DNA molecules.
In bioethics, there are a variety of ethical positions regarding the practice and possibilities of cloning. The use of embryonic stem cells, which can be produced through SCNT, in some stem cell research has attracted controversy. Cloning has been proposed as a means of De-extinction. In popular culture, the concept of cloning—particularly human cloning—is often depicted in science fiction; depictions commonly involve themes related to identity, the recreation of historical figures or extinct species, or cloning for exploitation (e.g. cloning soldiers for warfare).
Many plants are well known for natural cloning ability, including blueberry plants, Hazel trees, the Pando trees, the Kentucky coffeetree, Myrica, and the American sweetgum.
It also occurs accidentally in the case of identical twins, which are formed when a fertilized egg splits, creating two or more embryos that carry identical DNA.
Cloning of any DNA fragment essentially involves four steps
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 sensitisation 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.
A useful tissue culture technique used to clone distinct lineages of cell lines involves the use of cloning rings (cylinders). In this technique a single-cell suspension of cells that have been exposed to a 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 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.
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. 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. The embryo will then form a blastocyst which has the potential to form/become any cell in the body.
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. The oocyte will react to the somatic cell nucleus, the same way it would to a sperm cell's nucleus.
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. 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. This creates a one-cell embryo. The grouped somatic cell and egg cytoplasm are then introduced to an electrical current. 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.
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. 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. 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 tenShukman, David (14 January 2014) China cloning on an 'industrial scale' BBC News Science and Environment, Retrieved 10 April 2014 and in 2016, a Korean Company Sooam Biotech was reported to be producing 500 cloned embryos per day.
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.
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 ethics scrutiny and is generally treated as an entirely different kind of operation.
Many trees, , , and other perennials form clonal colony 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 viviparity grasses also form asexually, termed apomixis, resulting in clonal populations of genetically identical individuals.
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 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. If both embryos are successful, it gives rise to monozygotic (identical) twins.
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. 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 clones were frogs, no adult cloned frog has yet been produced from a somatic adult nucleus donor cell. this is a source that was used in List of animals that have been cloned
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 , DNA-protein complexes that protect the end of linear . 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.
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.BBC. 22 February 2008. BBC On This Day: 1997: Dolly the sheep is cloned
Recent advances in biotechnology allowed the modification of wolf clones to make them appear similar to Dire wolf by a company named Colossal Biosciences. "The company used a combination of gene-editing techniques and ancient DNA found in fossils to engineer the newborn pups." It's currently contested whether this can be considered a true dire wolf, but this shows that gene modification and possibly cloning in the future could advance. They produced three of the white "dire wolves" and scientists are more interested in how this can be used for endangered animals.
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, . 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.
Advocates support development of therapeutic cloning to generate tissues and whole organs to treat patients who otherwise cannot obtain transplants, to avoid the need for immunosuppressive drugs, and to stave off the effects of aging.de Grey, Aubrey; Rae, Michael (September 2007). Ending Aging: The Rejuvenation Breakthroughs that Could Reverse Human Aging in Our Lifetime. New York, NY: St. Martin's Press, 416 pp. . Advocates for reproductive cloning believe that parents who cannot otherwise procreate should have access to the technology. Times Higher Education. 10 August 2001 In the news: Antinori and Zavos
Opponents of cloning have concerns that technology is not yet developed enough to be safe and that it could be prone to abuse (leading to the generation of humans from whom organs and tissues would be harvested), as well as concerns about how cloned individuals could integrate with families and with society at large.McGee, Glenn (2000). The Perfect Baby: Parenthood in the New World of Cloning and Genetics. Lanham: Rowman & Littlefield. Cloning humans could lead to serious violations of human rights.
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.Bob Sullivan, Technology correspondent for NBC News. November 262003 Religions reveal little consensus on cloning – Health – Special Reports – Beyond Dolly: Human CloningBainbridge, William Sims (October 2003). "Religious Opposition to Cloning" . Journal of Evolution and Technology. 13. There is at least one religion, Raëlism, in which cloning plays a major role.
Contemporary work on this topic is concerned with the ethics, adequate regulation and issues of any cloning carried out by humans, not potentially by extraterrestrials (including in the future), and largely also not replication – also described as mind cloning
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 as safe by the US FDA, its use is opposed by groups concerned about food safety.
In practical terms, the inclusion of "licensing requirements for embryo research projects and fertility clinics, restrictions on the commodification of eggs and sperm, and measures to prevent proprietary interests from monopolizing access to stem cell lines" in international cloning regulations has been proposed, albeit e.g. effective oversight mechanisms or cloning requirements have not been described.
Engineers have proposed a "lunar ark" in 2021 – storing millions of seed, spore, sperm and egg samples from Earth's contemporary species in a network of lava tubes on the Moon as a genetic backup. Similar proposals have been made since at least 2008. These also include sending human customer DNA, and a proposal for "a lunar backup record of humanity" that includes genetic information by Avi Loeb et al.
In 2020, the San Diego Zoo began a number of projects in partnership with the conservation organization Revive & Restore and the ViaGen Pets to clone individuals of genetically-impoverished endangered species. A Przewalski's horse was cloned from preserved tissue of a stallion whose genes are absent in the surviving populations of the species, which descend from twenty individuals. The clone, named Kurt, had been born to a domestic surrogate mother, and was partnered with a natural-born Przewalski's mare in order to socialize him with the species' natural behavior before being introduced to the Zoo's breeding herd. In 2023, a second clone of the original stallion, named Ollie, was born; this marked the first instance of multiple living clones of a single individual of an endangered species being alive at the same time. Also in 2020, a clone named Elizabeth Ann was produced of a female black-footed ferret that had no living descendants. While Elizabeth Ann became sterile due to secondary health complications, a pair of additional clones of the same individual, named Antonia and Noreen, were born to distinct surrogate mothers, and Antonia successfully reproduced later in the year.
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.
Many such "de-extinction" projects are being championed by the non-profit Revive & Restore.
In 2022, scientists showed major limitations and the scale of challenge of genetic-editing-based de-extinction, suggesting resources spent on more comprehensive de-extinction projects such as of the woolly mammoth may currently not be well allocated and substantially limited. Their analyses "show that even when the extremely high-quality Norway brown rat (R. norvegicus) is used as a reference, nearly 5% of the genome sequence is unrecoverable, with 1,661 genes recovered at lower than 90% completeness, and 26 completely absent", complicated further by that "distribution of regions affected is not random, but for example, if 90% completeness is used as the cutoff, genes related to immune response and olfaction are excessively affected" due to which "a reconstructed Christmas Island rat would lack attributes likely critical to surviving in its natural or natural-like environment".
In a 2021 online session of the Russian Geographical Society, Russia's defense minister Sergei Shoigu mentioned using the DNA of 3,000-year-old Scythian warriors to potentially bring them back to life. The idea was described as absurd at least at this point in news reports and it was noted that Scythians likely weren't skilled warriors by default.
The idea of cloning Neanderthals or bringing them back to life in general is controversial but some scientists have stated that it may be possible in the future and have outlined several issues or problems with such as well as broad rationales for doing so.
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. 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.
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.
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.
A 2016 study indicated that once cloned animals survive 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.
The concept of cloning, particularly human cloning, has featured a wide variety of science fiction works. An early fictional depiction of cloning is Bokanovsky's Process which features in Aldous Huxley's 1931 dystopian novel Brave New World. The process is applied to fertilized human eggs in vitro, causing them to split into identical genetic copies of the original.Huxley, Aldous; "Brave New World and Brave New World Revisited"; p. 19; HarperPerennial, 2005. 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.
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), (2002), The Island (2005), Tales of the Abyss (2006), and Moon (2009) to comedies such as Woody Allen's 1973 film Sleeper.
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 . 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. Science fiction films such as The Matrix and Star Wars: Episode II – Attack of the Clones have featured scenes of human fetus being cultured on an industrial scale in mechanical tanks.
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. 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.
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 to get older so she can see if they have Samantha's brown eyes and her seriousness". The operation cost $50,000 through the pet cloning company ViaGen Pets.
In films such as Roger Spottiswoode's 2000 The 6th Day, which makes use of the trope of a "vast clandestine laboratory ... filled with row upon row of 'blank' human bodies kept floating in tanks of nutrient liquid or in suspended animation", clearly fear is to be incited. In Clark's view, the biotechnology is typically "given fantastic but visually arresting forms" while the science is either relegated to the background or fictionalised to suit a young audience. Genetic engineering methods are weakly represented in film; Michael Clark, writing for The Wellcome Trust, calls the portrayal of genetic engineering and biotechnology "seriously distorted"
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.
In the 2013 television series Orphan Black, cloning is used as a scientific study on the behavioral adaptation of the clones. 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.
The Norman Spinrad's satirical The Iron Dream, published in 1972, concludes with 300 seven-foot-tall, blond, super-intelligent all-male SS clones in suspended animation launched into space to begin the Hitler analog's galactic empire in the aftermath of a genocidal race war. (They had won a Pyrrhic victory, the subhumans' leader having as his last act detonated a doomsday weapon, specifically a cobalt bomb, irredeemably contaminated the gene pool).
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 . 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.
The 1977 film Star Wars was set against the backdrop of a historical conflict called the Clone Wars. The events of this war were not fully explored until the prequel films (2002) and (2005), which depict a space war waged by a massive army of heavily armoured 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.
The exploitation of human clones for dangerous and undesirable work was examined in the 2009 British science fiction film Moon. 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.
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 . 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.
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