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, [ 24 ]  fish (for example the  hammerhead shark [ 25 ] ),  Cape honeybees , [ 26 ]  and lizards including the  Komodo dragon [ 25 ]  and several  whiptails . 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 may also be called  reproductive cloning .


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 1924 he and his student,  Hilde Mangold , were the first to perform  somatic-cell nuclear transfer  using  amphibian  embryos – one of the first steps towards cloning. [ 27 ]


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. [ 28 ]  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  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. [ 29 ]  If both embryos are successful, it gives rise to  monozygotic (identical) twins .


Dolly , a  Finn-Dorset   ewe , was the first mammal to have been successfully cloned from an adult somatic cell. Dolly was formed by taking a cell from the udder of her 6-year-old biological mother. [ 30 ]  Dolly's embryo was created by taking the cell and inserting it into a sheep ovum. It took 435 attempts before an embryo was successful. [ 31 ]  The embryo was then placed inside a female sheep that went through a normal pregnancy. [ 32 ]  She was cloned at the  Roslin Institute  in Scotland by British scientists Sir  Ian Wilmut  and  Keith Campbell  and lived there from her birth in 1996 until her death in 2003 when she was six. She was born on 5 July 1996 but not announced to the world until 22 February 1997. [ 33 ]  Her  stuffed remains  were placed at Edinburgh's  Royal Museum , part of the  National Museums of Scotland . [ 34 ]


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. [ 35 ]  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. [ 36 ]


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. [ 37 ]


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. [ 38 ]


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


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. [ 85 ]


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 2024 [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. [ 86 ]


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, [ 87 ]  to avoid the need for  immunosuppressive drugs , [ 86 ]  and to stave off the effects of aging. [ 88 ]  Advocates for reproductive cloning believe that parents who cannot otherwise procreate should have access to the technology. [ 89 ]


Opponents of cloning have concerns that technology is not yet developed enough to be safe [ 90 ]  and that it could be prone to abuse (leading to the generation of humans from whom organs and tissues would be harvested), [ 91 ] [ 92 ]  as well as concerns about how cloned individuals could integrate with families and with society at large. [ 93 ] [ 94 ]  Cloning humans could lead to serious violations of  human rights . [ 95 ]


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. [ 96 ] [ 97 ]  There is at least one religion,  Raëlism , in which cloning plays a major role. [ 98 ] [ 99 ] [ 100 ]


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 [ 101 ] [ 102 ] [ 103 ] [ 104 ]  – of potential whole brain emulations.


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, [ 105 ] [ 106 ]  its use is opposed by groups concerned about food safety. [ 107 ] [ 108 ]


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. [ 109 ]


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 . [ 110 ] [ 111 ]  The best current cloning techniques have an average success rate of 9.4 percent [ 112 ]  (and as high as 25 percent [ 37 ] ) when working with familiar species such as mice, [ note 1 ]  while cloning wild animals is usually less than 1 percent successful. [ 115 ]


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. [ 110 ] [ 116 ] [ 117 ] [ 118 ]  This is also referred to as "Conservation cloning". [ 119 ] [ 120 ]


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. [ 121 ] [ 122 ] [ 123 ]  Similar proposals have been made since at least 2008. [ 124 ]  These also include sending human customer DNA, [ 125 ]  and a proposal for "a lunar backup record of humanity" that includes genetic information by  Avi Loeb  et al. [ 126 ]


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. [ 127 ]


Many such "De-extinction" projects are being championed by the non-profit  Revive & Restore . [ 128 ]


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. [ 129 ] [ 130 ]  The challenges are formidable. Extensively degraded DNA that may be suitable for sequencing may not be suitable for cloning; it would have to be synthetically reconstituted. In any case, with currently available technology, DNA alone is not suitable for mammalian cloning; intact viable cell nuclei are required.  Patching pieces of reconstituted mammoth DNA into an Asian elephant cell nucleus would result in an elephant-mammoth hybrid rather than a true mammoth. [ 131 ]  Moreover, true de-extinction of the wooly mammoth species would require a breeding population, which would require cloning of multiple genetically distinct but reproductively compatible individuals, multiplying both the amount of work and the uncertainties involved in the project.  There are potentially other post-cloning problems associated with the survival of a reconstructed mammoth, such as the requirement of   ruminants  for specific  symbiotic   microbiota  in their stomachs for digestion. [ 131 ]


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". [ 132 ]


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. [ 133 ] [ 134 ] [ 135 ]


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. [ 136 ] [ 137 ] [ 138 ] [ 139 ] [ 140 ] [ 141 ]


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 . [ 142 ] [ 143 ] [ 144 ] [ 145 ]


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 . [ 146 ]  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 . [ 147 ]


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. [ 148 ]


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. [ 37 ] [ 149 ]  Other sources have noted that the offspring of clones tend to be healthier than the original clones and indistinguishable from animals produced naturally. [ 150 ]


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. [ 151 ] [ dubious  –  discuss ]


A 2016 study indicated that once cloned animals survive the first month or two of life they are generally healthy. [ 152 ]  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. [ 38 ]


Discussion of cloning in the popular media often presents the subject negatively. In an article in the 8 November 1993 article of  Time , cloning was portrayed in a negative way, modifying Michelangelo's  Creation of Adam  to depict Adam with five identical hands. [ 153 ]   Newsweek 's  10 March 1997 issue also critiqued the ethics of human cloning, and included a graphic depicting identical babies in beakers. [ 154 ]


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. [ 155 ] [ 156 ]  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. [ 157 ]


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),  Tales of the Abyss  (2006), and  Moon  (2009) to comedies such as  Woody Allen 's 1973 film  Sleeper . [ 158 ]


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. [ 159 ]  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. [ 160 ]


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. [ 161 ]  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. [ 162 ]


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". [ 163 ]  The operation cost $50,000 through the pet cloning company  ViaGen . [ 164 ]


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. [ 165 ]  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" [ 165 ]


Science fiction has used cloning, most commonly and specifically  human cloning , to raise questions of identity. [ 166 ] [ 167 ]   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 . [ 168 ]


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. [ 169 ]


In the 2013 television series  Orphan Black , cloning is used as a scientific study on the behavioral adaptation of the clones. [ 170 ]  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. [ 171 ]


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 . [ 172 ]


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 . [ 173 ]


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. [ 174 ]  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 . [ 175 ]


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  Attack of the Clones  (2002) and  Revenge of the Sith  (2005), which depict a  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. [ 176 ] [ 177 ]


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 [ 178 ]  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 [ 179 ]  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 . [ 180 ]  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. [ 181 ]


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. [ 182 ] [ 183 ]


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  tissue . It does not refer to the natural conception and delivery of  identical   twins . The possibilities of human cloning  have raised controversies . These ethical concerns have prompted several nations to pass laws regarding human cloning.


Two commonly discussed types of human cloning are  therapeutic cloning  and  reproductive cloning .


Therapeutic cloning would involve cloning cells from a human for use in medicine and transplants. It is an active area of research, and is in medical practice over 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.


Although the possibility of  cloning  humans had been the subject of speculation for much of the 20th century, scientists and policymakers began to take the prospect seriously in 1969.  J. B. S. Haldane  was the first to introduce the idea of human cloning, for which he used the terms "clone" and "cloning", [ 1 ]  which had been used in agriculture since the early 20th century. In his speech on "Biological Possibilities for the Human Species of the Next Ten Thousand Years" at the  Ciba Foundation  Symposium on Man and his Future  in 1963, he said: [ 2 ]


It is extremely hopeful that some human cell lines can be grown on a medium of precisely known chemical composition. Perhaps the first step will be the production of a clone from a single fertilized egg, as in  Brave New World ...
Assuming that cloning is possible, I expect that most clones would be made from people aged at least fifty, except for athletes and dancers, who would be cloned younger. They would be made from people who were held to have excelled in a socially acceptable accomplishment...


Nobel Prize-winning geneticist  Joshua Lederberg  advocated cloning and  genetic engineering  in an article in  The American Naturalist  in 1966 and again, the following year, in  The Washington Post . [ 3 ]  He sparked a debate with conservative bioethicist  Leon Kass , who wrote at the time that "the programmed reproduction of man will, in fact, dehumanize him." Another  Nobel Laureate ,  James D. Watson , publicized the potential and the perils of cloning in his  Atlantic Monthly  essay, "Moving Toward the Clonal Man", in 1971. [ 4 ]


With the cloning of a sheep known as  Dolly  in 1996 by  somatic cell nuclear transfer  (SCNT), the idea of human cloning became a hot debate topic. [ 5 ]  Many nations outlawed it, while a few scientists promised to make a clone within the next few years. The first  hybrid  human clone was created in November 1998, by  Advanced Cell Technology . It was created using SCNT; a nucleus was taken from a man's leg cell and inserted into a cow's egg from which the nucleus had been removed, and the hybrid cell was cultured and developed into an  embryo . The embryo was destroyed after 12 days. [ 6 ]


In 2004 and 2005,  Hwang Woo-suk , a professor at  Seoul National University , published two separate articles in the journal   Science  claiming to have successfully harvested pluripotent,  embryonic stem cells  from a cloned human blastocyst using SCNT techniques. Hwang claimed to have created eleven different patient-specific stem cell lines. This would have been the first major breakthrough in human cloning. [ 7 ]  However, in 2006  Science  retracted both of his articles on account of clear evidence that much of his data from the experiments was fabricated. [ 8 ]


In January 2008, Dr. Andrew French and  Samuel Wood  of the biotechnology company  Stemagen  announced that they successfully created the first five mature human embryos using SCNT. In this case, each embryo was created by taking a nucleus from a skin cell (donated by Wood and a colleague) and inserting it into a human egg from which the nucleus had been removed. The embryos were developed only to the  blastocyst  stage, at which point they were studied in processes that destroyed them.  Members of the lab said that their next set of experiments would aim to generate embryonic stem cell lines; these are the "holy grail" that would be useful for therapeutic or reproductive cloning. [ 9 ] [ 10 ]


In 2011, scientists at the New York Stem Cell Foundation announced that they had succeeded in generating embryonic stem cell lines, but their process involved leaving the  oocyte 's nucleus in place, resulting in  triploid  cells, which would not be useful for cloning. [ 11 ] [ 12 ] [ 13 ]


In 2013, a group of scientists led by  Shoukhrat Mitalipov  published the first report of embryonic stem cells created using SCNT. In this experiment, the researchers developed a protocol for using SCNT in human cells, which differs slightly from the one used in other organisms.  Four embryonic stem cell lines from human fetal somatic cells were derived from those blastocysts. All four lines were derived using oocytes from the same donor, ensuring that all  mitochondrial DNA  inherited was identical. [ 11 ]  A year later, a team led by  Robert Lanza  at Advanced Cell Technology reported that they had replicated Mitalipov's results and further demonstrated the effectiveness by cloning adult cells using SCNT. [ 5 ] [ 14 ]


In 2018, the first successful  cloning  of  primates  using SCNT was reported with the birth of two live female clones,  crab-eating macaques  named  Zhong Zhong and Hua Hua . [ 15 ] [ 16 ]


In somatic cell nuclear transfer ("SCNT"), the nucleus of a  somatic cell  is taken from a donor and transplanted into a host  egg cell , which had its own genetic material removed previously, making it an enucleated egg. After the donor somatic cell genetic material is transferred into the host oocyte with a micropipette, the somatic cell genetic material is fused with the egg using an electric current. Once the two cells have fused, the new cell can be permitted to grow in a  surrogate  or  artificially . [ 17 ]  This is the process that was used to successfully clone Dolly the sheep (see  § History ). [ 5 ]  The technique, now refined, has indicated that it was possible to replicate cells and reestablish pluripotency, or "the potential of an embryonic cell to grow into any one of the numerous different types of mature body cells that make up a complete organism". [ 18 ]


Creating induced  pluripotent   stem cells  ("iPSCs") is a long and inefficient process.  Pluripotency  refers to a stem cell that has the potential to  differentiate  into any of the three  germ layers :  endoderm  (interior stomach lining, gastrointestinal tract, the lungs),  mesoderm  (muscle, bone, blood, urogenital), or  ectoderm  (epidermal tissues and nervous tissue). [ 19 ]  A specific set of genes, often called "reprogramming factors", are introduced into a specific adult cell type. These factors send  signals  in the mature cell that cause the cell to become a pluripotent stem cell. This process is highly studied and new techniques are being discovered frequently on how to improve this induction process.


Depending on the method used, reprogramming of adult cells into iPSCs for  implantation  could have severe limitations in humans. If a virus is used as a reprogramming factor for the cell, cancer-causing genes called  oncogenes  may be  activated . These cells would appear as rapidly dividing cancer cells that do not respond to the body's natural cell signaling process. However, in 2008 scientists discovered a technique that could remove the presence of these oncogenes after pluripotency induction, thereby increasing the potential use of iPSC in humans. [ 20 ]


Both the processes of SCNT and iPSCs have benefits and deficiencies. Historically, reprogramming methods were better studied than SCNT derived embryonic stem cells (ESCs). [ 11 ]  However, more recent studies have put more emphasis on developing new procedures for SCNT-ESCs. The major advantage of SCNT over iPSCs at this time is the speed with which cells can be produced. iPSCs derivation takes several months while SCNT would take a much shorter time, which could be important for medical applications. New studies are working to improve the process of iPSC in terms of both speed and efficiency with the discovery of new reprogramming factors in oocytes. [ citation needed ]  Another advantage SCNT could have over iPSCs is its potential to treat  mitochondrial disease , as it uses a donor oocyte. [ 11 ]  No other advantages are known at this time in using stem cells derived from one method over stem cells derived from the other. [ 21 ]


Work on cloning techniques has advanced understanding of  developmental biology  in humans. Observing human pluripotent stem cells grown in culture provides great insight into  human embryo development , which otherwise cannot be seen. Scientists are now able to better define steps of early human development. Studying  signal transduction  along with genetic manipulation within the early human embryo has the potential to provide answers to many developmental diseases and defects. Many human-specific signaling pathways have been discovered by studying human embryonic stem cells. Studying developmental pathways in humans has given developmental biologists more evidence toward the hypothesis that developmental pathways are conserved throughout species. [ 22 ]


iPSCs and cells created by SCNT are useful for research into the causes of disease, and as model systems used in  drug discovery . [ 23 ] [ 24 ]


Cells produced with SCNT, or iPSCs could eventually be used in  stem cell therapy , [ 25 ]  or to  create organs  to be used in transplantation, known as  regenerative medicine . Stem cell therapy is the use of stem cells to treat or prevent a disease or condition.  Bone marrow transplantation  is a widely used form of stem cell therapy. [ 26 ]  No other forms of stem cell therapy are in clinical use at this time. Research is underway to potentially use stem cell therapy to treat  heart disease ,  diabetes , and  spinal cord injuries . [ 27 ] [ 28 ]  Regenerative medicine is not in clinical practice, but is heavily researched for its potential uses. This type of medicine would allow for autologous transplantation, thus removing the risk of organ transplant rejection by the recipient. [ 29 ]  For instance, a person with liver disease could potentially have a new liver grown using their same genetic material and transplanted to remove the damaged liver. [ 30 ]  In current research, human pluripotent stem cells have been promised as a reliable source for generating human neurons, showing the potential for regenerative medicine in brain and neural injuries. [ 31 ]


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, for instance relating to Christian views of procreation and personhood, [ 32 ]  the questions raised by cloning engage secular perspectives as well, particularly the concept of identity. [ 33 ]


Advocates support development of therapeutic cloning in order to generate tissues and whole organs to treat patients who otherwise cannot obtain transplants, [ 34 ]  to avoid the need for  immunosuppressive drugs , [ 35 ]  and to stave off the effects of aging. [ 36 ]   Advocates for reproductive cloning believe that parents who cannot otherwise procreate should have access to the technology. [ 37 ]


Opposition to therapeutic cloning mainly centers around the status of  embryonic stem cells , which has connections with the  abortion debate . [ 38 ]  The moral argument put forward is based on the notion that embryos deserve protection from the moment of their conception because it is at this precise moment that a new human entity emerges, already a unique individual. [ 39 ]  Since it is deemed unacceptable to sacrifice human lives for any purpose, the argument asserts that the destruction of embryos for research purposes is no longer justifiable. [ 40 ]


Some opponents of reproductive cloning have concerns that technology is not yet developed enough to be safe – for example, the position of the  American Association for the Advancement of Science  as of 2014, [update] [ 41 ]  while others emphasize that reproductive cloning could be prone to abuse (leading to the generation of humans whose organs and tissues would be harvested), [ 42 ] [ 43 ]  and have concerns about how cloned individuals could integrate with families and with society at large. [ 44 ] [ 45 ]


Members of religious groups are divided. Some Christian theologians perceive the technology as usurping God's role in creation and, to the extent embryos are used, destroying a human life; [ 32 ]  others see no inconsistency between Christian tenets and cloning's positive and potentially life-saving benefits. [ 46 ] [ 47 ]


There have been consistent calls in Canada to ban human reproductive cloning since the 1993 Report of the Royal Commission on New Reproductive Technologies. Polls have indicated that an overwhelming majority of Canadians oppose human reproductive cloning, though the regulation of human cloning continues to be a significant national and international policy issue.  The notion of "human dignity" is commonly used to justify cloning laws. The basis for this justification is that reproductive human cloning necessarily infringes notions of human dignity. [ 57 ] [ 58 ] [ 59 ] [ 60 ]


In the Eleventh Amendment to the Criminal Law, which came into effect on March 1, 2021, an additional provision was added to Article 336, which stipulates that "implanting gene-edited or cloned human embryos into human or animal bodies, or implanting gene-edited, cloned Implantation of cloned animal embryos into human bodies, if the circumstances are serious, shall be sentenced to fixed-term imprisonment of not more than three years or criminal detention and a fine; if the circumstances are especially serious, the sentence shall be fixed-term imprisonment of not less than three years but not more than seven years and a fine." [ 62 ]


Albania ,  Andorra ,  Bosnia and Herzegovina ,  Bulgaria ,  Croatia ,  Cyprus ,  Czech Republic ,  Denmark ,  Estonia ,  Finland ,  France ,  Georgia ,  Greece ,  Hungary ,  Iceland ,  Latvia ,  Liechtenstein ,  Lithuania ,  Moldova ,  Montenegro ,  North Macedonia ,  Norway ,  Portugal ,  Romania ,  San Marino ,  Serbia ,  Slovakia ,  Slovenia ,  Spain ,  Switzerland ,  Turkey


India has already succeeded in mammalian cloning. [ 75 ]


In Morocco, all research on human embryos or fetuses is forbidden, as is the conception of human embryos or fetuses for research or experimental purposes, in accordance with article 7 of Dahir no. 1–19–50. [ 78 ]


The first license was granted on 11 August 2004, to researchers at the  University of Newcastle  to allow them to investigate treatments for  diabetes ,  Parkinson's disease  and  Alzheimer's disease . [ 92 ]  The  Human Fertilisation and Embryology Act 2008 , a major review of fertility legislation, repealed the 2001 Cloning Act by making amendments of similar effect to the 1990 Act. The 2008 Act also allows experiments on hybrid human-animal embryos. [ 93 ]


In 1998, 2001, 2004, 2005, 2007 and 2009, the United States Congress voted whether to ban all human cloning, both reproductive and therapeutic ( Stem Cell Research Enhancement Act ). [ 98 ]  Divisions in the  Senate , or an eventual veto from the sitting President ( George W. Bush  in 2005 and 2007), over therapeutic cloning prevented either competing proposal (a ban on both forms or on reproductive cloning only) from being passed into law. On 10 March 2010, a bill (HR 4808) was introduced with a section banning federal funding for human cloning. [ 99 ]  Such a law, if passed, would not have prevented research from occurring in private institutions (such as universities) that have both private and federal funding. However, the 2010 law was not passed.


Ten states, California, Connecticut, Illinois, Iowa, Maryland, Massachusetts, Missouri, Montana, New Jersey and Rhode Island, have "clone and kill" laws that prevent cloned embryo implantation for childbirth, but allow embryos to be destroyed. [ 100 ]


The Patients First Act of 2017 (HR 2918, 115th Congress) aims to promote stem cell research, using cells that are "ethically obtained", that could contribute to a better understanding of diseases and therapies, as well as promote the "derivation of pluripotent stem cell lines without the creation of human embryos". [ 101 ]


Science fiction has used cloning, most commonly and specifically human cloning, due to the fact that it brings up controversial questions of identity. [ 111 ] [ 112 ]  Humorous fiction, such as  Multiplicity  (1996) [ 113 ]  and the Maxwell Smart feature  The Nude Bomb  (1980), have featured human cloning. [ 114 ]  A recurring sub-theme of cloning fiction is the use of clones as a supply of  organs  for  transplantation . Robin Cook's 1997 novel  Chromosome 6 , Michael Bay's  The Island , and Nancy Farmer's 2002 novel  House of the Scorpion [ 115 ]  are examples of this;  Chromosome 6  also features  genetic manipulation  and  xenotransplantation . [ 116 ]  The  Star Wars  saga makes use of millions of human clones to form the  Grand Army of the Republic  that participated in the  Clone Wars . The series  Orphan Black  follows human clones' stories and experiences as they deal with issues and react to being the property of a chain of scientific institutions. [ 117 ]  In the 2019 horror film  Us , the entirety of the United States' population is secretly cloned. Years later, these clones (known as The Tethered) reveal themselves to the world by successfully pulling off a mass genocide of their counterparts. [ 118 ] [ 119 ]


In the 2005 novel  Never Let Me Go ,  Kazuo Ishiguro  crafts a subtle exploration into the ethical complications of cloning humans for medical advancement and longevity.


In the field of  cell biology , the method of  partial cloning  ( PCL ) converts a fully differentiated  old   somatic cell  into a  partially reprogrammed   young  cell that retains all the specialised functions of the differentiated  old  cell but is simply younger. [ 1 ]   The method of PCL reverses characteristics associated with old cells.  For example, old, senescent, cells  rejuvenated  by PCL are free of highly condensed senescence-associated heterochromatin foci (SAHF) and re-acquire the proliferation potential of young cells. [ 2 ]   The method of PCL thus rejuvenates old cells without de-differentiation and passage through an embryonic, pluripotent, stage.


PCL consists in introducing a  somatic  adult or senescent  cell nucleus  or entire cell with enlarged membrane pores in an (activated)  oocyte  and to withdraw this treated cell before its de-differentiation and first  cell division  occurs. Thus, the progressive rejuvenation capability of the oocyte is used only temporarily in order to obtain a partial natural rejuvenation. PCL permits to envisage a chosen degree of partial rejuvenation in changing the duration of the introduction of the treated cell in the oocyte. Using PCL cell de-differentiation and its age reprogramming might be, at least partially, separable. Thus the existence of an isolated ageing clock would be confirmed at least during a certain part of the  cellular evolution  and  involution .


First experimental result shows a possible high efficiency in partial rejuvenation of senescent  mouse  cells. Notably PCL rejuvenates exclusively one single tissue or organ, in contrast to classical  cloning  PCL is therefore unable to reconstitute an entire organism. Furthermore, PCL is feasible in a few hours in opposition to classical cloning or  induced pluripotent stem cells  (iPS) which all need weeks or months.


Classical cloning can rejuvenate old cells but the process demands that the old cells must artificially pass through an embryonic cell stage. Partial cloning affords the advantage that the old cells to be rejuvenated do not have to pass through the embryonic cell stage and are simply made younger.


The extension of human lifespan, in terms of useful, quality, years added to life, has been a goal for many since time immemorial. And while a goal whose attainment was thought improbable, or at least achievable only in the far distant future, the discovery that animals can be cloned has brought the goal of rejuvenation much closer. The remarkable discovery that animals can be cloned showed that the nucleus of an old cell can be used as a donor in so-called “nuclear transfer” experiments where an old nucleus is transferred into a recipient egg whose own nuclear material has been removed. The “reconstructed” egg is then prompted to engage development and develops through an embryonic stage that results, once the embryo is implanted into a surrogate mother, into a new born.  Thus an old cell can give rise to a newborn, which has a typical lifespan: the age of the donor cell is “wiped clean” and returned to a youthful state. Notably, in classical animal cloning the rejuvenation process involves a return to an embryonic form. Thus the specialized functions of the adult cell are also “wiped clean” and returned to an embryonic cell type. And in classical cloning passage through this embryonic state is a must for the age of the cell to be “wiped clean”.


The key notion that exemplifies “partial” cloning from “classical” cloning is the separation of the mechanism(s) that “wipe clean” the specialization of a cell from those that “wipe-clean” the age of the cell. In short, partial cloning aims to retain the specialized functions of a cell and simply make it younger, e.g., a skin cell is rejuvenated without having to pass through the embryonic stage that is a must for rejuvenation via the classical cloning technique (see diagram).


In a new laboratory at the Forschungszentrum Borstel our work on partial cloning focuses, inter alia, on the restricted, temporary, incubation of an “old” cell within the egg. In this way only the age of the cell is “wiped clean” and its specialized, differentiated, state is retained. It is simply made younger – rejuvenated - without going through the embryonic state. The measure of Diagram showing the difference between “Classical” and “Partial” cloning: Classical cloning (the route given by the black arrows) can rejuvenate an old cell but requires passage through an embryonic stage. “Partial cloning” (given by the red arrow) rejuvenates old cells without passage through an embryonic stage.“Partial cloning” (given by the red arrow) rejuvenates old cells without passage through an embryonic stage.
In a new laboratory at the Forschungszentrum Borstel our work on partial cloning focuses, inter alia, on the restricted, temporary, incubation of an “old” cell within the egg. In this way only the age of the cell is “wiped clean” and its specialized, differentiated, state is retained. It is simply made younger – rejuvenated - without going through the embryonic state. The measure of rejuvenation in our system is, first, the re-acquisition of the ability of an old cell to divide, something that is lost in old cells and, second, the loss of characteristics that are associated with old cells.


Should such rejuvenation be achievable the consequences for medicine would be profound. It would avoid the need to artificially pass through an embryonic stage – either by nuclear transfer or by the so-called iPS cells method - to rejuvenate cells. One would simply be able to take aged cells from a patient and then return to the patient their own, histocompatible, rejuvenated heart cells, liver cells etc. In sharp contrast to the cycle of artificial de-differentiation of somatic cells to stem cells and then the artificial re-differentiation of stem cells to the desired differentiated cell type, which is highly inefficient, time-consuming and results in unstable cell types. The process of partial cloning would be efficient and rapid and thus cheap both in terms of materials and manpower. In short, partial cloning has enormous potential to relieve human suffering and disease: it is the most rapid and cheap route to successful regenerative medicine. Partial cloning also avoids the ethical problems associated with “classical” cloning in that it does not result in live born – it mere uses the oocyte briefly as a means to condition and thereby rejuvenate the old cell exclusively.


Artificial reproduction  is the re-creation of life brought about by means other than  natural ones.  It is new life built by human plans and projects. Examples include  artificial selection ,  artificial insemination ,  in vitro fertilization ,  artificial womb , artificial  cloning , and  kinematic replication .


Artificial reproduction is one aspect of  artificial life . Artificial reproduction can be categorized into one of two classes according to its capacity to be  self-sufficient : non-assisted reproductive technology and  assisted reproductive technology .


Cutting  plants' stems and placing them in  compost  is a form of  assisted  artificial reproduction,  xenobots  are an example of a more autonomous type of reproduction, while the artificial womb presented in the movie  the Matrix  illustrates a  non assisted   hypothetical technology . The idea of artificial reproduction has led to various  technologies .


Humans have aspired to create life since immemorial times. Most  theologies  and religions have conceived this possibility as exclusive of deities. Christian religions consider the possibility of artificial reproduction, in most cases, as heretical and sinful.


Although  ancient Greek philosophy  raised the concept that man could imitate the creative capacity of nature, classic Greeks thought that if possible, human beings would reproduce things as nature does, and vice versa, nature would do the things that man does in the same way.  Aristotle , for example, wrote that if nature made tables, it would make them just as men do. In other words, Aristotle said that if nature were to create a table, such table will look like a human-made table. Correspondingly,  Descartes  envisioned the human body, and nature, as a machine. Cartesian philosophy does not stop seeing a perfect mirror between nature and the artificial.


However,  Kant  revolutionized this old idea by criticizing such  naturalism . Kant pedagogically wrote:


"Reason, in order to be taught by nature, must approach nature with its principles in one hand, according to which the agreement among appearances can count as laws, and, in the other hand, the experiment thought out in accord with these principles—in order to be instructed by nature not like a pupil, who has recited to him whatever the teacher wants to say, but like an appointed judge who compels witnesses to answer the questions he puts to them.".


Humans are not instructed by nature but rather use nature as raw material to invent. Humans find alternatives to the natural restrictions imposed by natural laws thus, nature is not necessarily mirrored. In accordance with Kant (and contrary to what Aristotle thought)  Karl Marx ,  Alfred Whitehead , [ 1 ]   Jaques Derrida  and  Juan David García Bacca  noticed that nature is incapable of reproducing tables; or airplanes, or submarines, or computers. [ 2 ]  If nature tried to create airplanes, it would produce birds. If nature tried to create submarines, it would get fishes. If nature tried to create computers, brains would grow. And if nature tried to create man, modern man, monkeys will be evolved. According to Whitehead, if we look for something natural in artificial life, in the most elaborate cases, if anything, only atoms remain natural.
Juan David Garcia Bacca summarized,


“It will not come out from wood, it will not be born, a galley; from clay, a vessel; from linen, a dress; from iron, a lever,...From natural, artificial. In the artificial, the natural is reduced to a simple raw material, even though it is perfectly specified with natural specification. The artificial is the real, positive, and original negation of the natural: of species, of genus and of essence. Thus, its ontology is superior to natural ontology. And for this very reason Marx did not attach any importance to Darwin, whose evolutionism is confined to the natural order: to changes, at most, from variety to variety, from species to species... natural. For the same reason, nature has no dialectics, even though continuous evolution and selection can occur. The dialectic cannot emerge from the natural, for deeper reasons than, using today's terms, from a bird, an airplane cannot emerge; from fish, a submarine; from ears, a telephone; from eyes, a television; from a brain, a digital computer; from feet, a car; from hands, an engine; from Euclid,
Descartes; from Aristotle, Newton; from Plato, Marx.”


According to García Bacca, the major difference between  natural causes  and  artificial causes  is that nature does not have plans and projects, while humans design things following plans and projects.


In contrast, other influential authors such as  Michael Behe  have depicted the concept and promoted the idea of  intelligent design , a notion that has aroused several doubts and heated controversies, as it reframe natural causes in accordance with a natural plan. Previous ideas that have also provided a positive 'sense' to natural reproduction, are  orthogenesis ,  syntropy ,  orgone  and  morphic resonance , among others. Although, these ideas have been historically marginalized and often called  pseudoscience , recently  Bio-semioticians  are reconsidering some of them under symbolic approaches.


Current metaphysics of science actually recognizes that the artificial ways of reproduction are diverse from nature, i.e., unnatural, anti-natural or supernatural. Because  Biosemiotics  does not focus on the  function  of life but on its  meaning , it has a better understanding of the artificial than classic biology.


Biology , being the study of cellular life, addresses reproduction in terms of growth and cellular division (i.e., binary fission,  mitosis and meiosis); however, the science of artificial reproduction is not restricted by the mirroring of these natural processes.The science of artificial reproduction is actually transcending the natural forms, and natural rules, of reproduction.  For example,  xenobots have redefined the classical conception of reproduction. Although xenobots are made of eukariotic cells they do not reproduce by mitosis, but rather by kinematic replication. Such constructive replication does not involve  growing  but rather  building .


Assisted reproductive technology  ( ART )'s purpose is to assist the development of a human embryo, commonly because of medical concerns due to  fertility limitations.


Non-assisted reproductive technologies  (NART) could have medical motivations but are mostly driven by a wider  heterotopic  ambition. Although, NARTs are initially designed by humans, they are programed to become independent of humans to a relative or absolute extent.  James Lovelock  proposed that such novelties could overcome humans. [ 3 ]


Cloning is the cellular reproductive processes where two or more genetically identical organisms are created, either by natural or artificial means.  Artificial cloning  normally involves editing the genetic code, somatic cell nuclear transfer and  3D bioprinting .


A non-assisted  artificial womb  or artificial uterus is a device that allow for  ectogenesis  or extracorporeal pregnancy by growing an embryonic form outside the body of an organism (that would normally carry the embryo to term) without any human assistance. The aspect of non-assistance is the key distinction between the current artificial womb technology (AWT) in modern medical research, which still relies on human assistance. With this non-assisted hypothetical technology, a  zygote  or  stem cells  are used to create an embryo that is then incubated and monitored by  artificial intelligence  (AI) within a chamber composed of biocompatible material. The AI maintains the necessary conditions for the embryo to develop and thrive, proceeding to mimic organic labor and childbirth in order to best help the embryo adjust to the outside world. [ 4 ] [ 5 ]


Ectogenesis—gestation, depicted in the science fiction movie The Matrix, is a fast approaching reality. [ citation needed ]  This type of innovation presupposes that vertebrate wombs are not the only way for bearing humans or other similar forms of life. [ 6 ]


Self-replication without binary fission, meiosis, mitosis (or any other form of cellular reproduction that involves division and growing) can be achieved.  Xenobots  are an example of kinematic replication. [ 7 ]  They are biobots, named after the African clawed frog ( Xenopus laevis ). Xenobots are cellular life forms designed by using artificial intelligence to build more of themselves by combining frog cells in a liquid medium. [ 8 ] [ 9 ] [ 10 ] [ 11 ] [ 12 ] [ 13 ]


The term kinematic replication is usually reserved for biomolecules (e.g. DNA, RNA,  prions , etc.) and artificially designed cellular forms (e.g. xenobots).


Machine constructive replication mimics human traditional manufacturing but is entirely self-automated. Such constructive replication is a more general form of kinematic replication, which does not necessarily includes bio-molecular or cellular forms. This technology also includes non-organic forms of life such as robots, cyborgs and artificial intelligence reproduction. [ 14 ]  Constructive replication, as kinematic  replication, does not involve growing. [ 15 ]  In nature growing is required for cellular reproduction, where a cell grows before it splits in two daughters cells. Examples of cellular division are  binary fission ,  mitosis  and  meiosis ; these natural reproductive processes require growing, however constructive replication does not require growing but rather a non-human subject performing the construction of more of itself by using available raw materials.


In computational terms, constructive replication is understood as a multi-step process which involves self-learning algorithms to assemble machines, [ 16 ]  and it could involve machines collecting resources. [ 17 ]  Each machine is created with a neural-network "brain" that can learn and adapt based on information it gathers. That machine's goal is then to manufacture more of itself in the best way it can come up with.


Such automated constructive replication involves the notion of  inheritance  and learning tasks, as machines create an exact copy of themselves through a blueprint that has been passed on to them. [ 18 ]  Each machine then learns over time, making modifications to its software and its blueprint for future machines' hardware. It then passes on that modified blueprint to the machines it creates or helps create.


Amplification of an existing  consciousness  is a  hypothetical technology . [ 19 ]  This idea has inspired several movies,  Chappie (film)  and  Detroit: Become Human . [ citation needed ]  The reproduction of  AI  is currently part of an innovative human project, [ 20 ]  involving code and the amplification of that code. In other terms, the reproduction could come from information the AI collected across the Internet.


Blastulation  is the stage in early  animal   embryonic development  that produces the  blastula . In mammalian development, the blastula develops into the  blastocyst  with a differentiated  inner cell mass  and an outer  trophectoderm . The blastula (from Greek  βλαστός  ( blastos  meaning  sprout )) is a hollow sphere of  cells  known as  blastomeres  surrounding an inner fluid-filled cavity called the  blastocoel . [ 1 ] [ 2 ]  Embryonic development begins with a  sperm   fertilizing  an  egg cell  to become a  zygote , which undergoes many  cleavages  to develop into a ball of cells called a  morula . Only when the blastocoel is formed does the early embryo become a blastula. The blastula precedes the formation of the  gastrula  in which the  germ layers  of the embryo form. [ 3 ]


A common feature of a  vertebrate  blastula is that it consists of a layer of blastomeres, known as the  blastoderm , which surrounds the blastocoel. [ 4 ] [ 5 ]  In  mammals , the blastocyst contains an  embryoblast  (or inner cell mass) that will eventually give rise to the definitive structures of the  fetus , and a  trophoblast  which goes on to form the extra-embryonic tissues. [ 3 ] [ 6 ]


During blastulation, a significant amount of activity occurs within the early embryo to establish  cell polarity ,  cell specification ,  axis formation , and to regulate  gene expression . [ 7 ]  In many animals, such as  Drosophila  and  Xenopus , the  mid blastula transition  (MBT) is a crucial step in development during which the  maternal mRNA  is degraded and control over development is passed to the embryo. [ 8 ]  Many of the interactions between blastomeres are dependent on  cadherin  expression, particularly  E-cadherin  in mammals and EP-cadherin in  amphibians . [ 7 ]


The study of the blastula, and of cell specification has many implications in  stem cell research , and  assisted reproductive technology . [ 6 ]  In  Xenopus , blastomeres behave as  pluripotent  stem cells which can migrate down several pathways, depending on  cell signaling . [ 9 ]  By manipulating the cell signals during the blastula stage of development, various  tissues  can be formed. This potential can be instrumental in  regenerative medicine  for disease and injury cases.  In vitro fertilisation  involves the  transfer of an embryo  into a  uterus  for  implantation . [ 10 ]


The blastula stage of early embryo development begins with the appearance of the blastocoel. The origin of the blastocoel in  Xenopus  has been shown to be from the first  cleavage furrow , which is widened and sealed with  tight junctions  to  create a cavity . [ 11 ]


In many organisms the development of the embryo up to this point and for the early part of the blastula stage is controlled by maternal mRNA, so called because it was produced in the egg prior to fertilization and is therefore exclusively from the mother. [ 12 ] [ 13 ]


In many organisms including  Xenopus  and  Drosophila , the  midblastula transition  usually occurs after a particular number of cell divisions for a given species, and is defined by the ending of the synchronous cell division cycles of the early blastula development, and the lengthening of the  cell cycles  by the addition of the  G1  and  G2 phases .  Prior to this transition, cleavage occurs with only the synthesis and mitosis phases of the cell cycle. [ 13 ]  The addition of the two growth phases into the cell cycle allows for the cells to increase in size, as up to this point the blastomeres undergo reductive divisions in which the overall size of the embryo does not increase, but more cells are created. This transition begins the growth in size of the organism. [ 3 ]


The mid-blastula transition is also characterized by a marked increase in  transcription  of new, non-maternal mRNA transcribed from the genome of the organism. Large amounts of the maternal mRNA are destroyed at this point, either by proteins such as  SMAUG  in  Drosophila [ 14 ]  or by  microRNA . [ 15 ]  These two processes shift the control of the embryo from the maternal mRNA to the nuclei.


A blastula ( blastocyst  in  mammals ), is a sphere of cells surrounding a fluid-filled cavity called the  blastocoel . The blastocoel contains  amino acids ,  proteins ,  growth factors , sugars, ions and other components which are necessary for  cellular differentiation . The blastocoel also allows blastomeres to move during the process of  gastrulation . [ 16 ]


In  Xenopus  embryos, the blastula is composed of three different regions. The animal cap forms the roof of the blastocoel and goes on primarily to form  ectodermal  derivatives. The equatorial or marginal zone, which compose the walls of the blastocoel differentiate primarily into  mesodermal  tissue. The vegetal mass is composed of the blastocoel floor and primarily develops into  endodermal  tissue. [ 7 ]


In the mammalian blastocyst there are three lineages that give rise to later tissue development. The  epiblast  gives rise to the fetus itself while the trophoblast develops into part of the  placenta  and the primitive endoderm becomes the  yolk sac . [ 6 ]  In the mouse embryo, blastocoel formation begins at the 32-cell stage. During this process, water enters the embryo, aided by an osmotic gradient which is the result of  sodium–potassium pumps  that produce a high sodium gradient on the basolateral side of the trophectoderm. This movement of water is facilitated by  aquaporins .  A seal is created by tight junctions of the  epithelial cells  that line the blastocoel. [ 6 ]


Tight junctions are very important in embryo development. In the blastula, these cadherin mediated cell interactions are essential to development of epithelium which are most important to  paracellular transport , maintenance of cell polarity and the creation of a permeability seal to regulate blastocoel formation. These tight junctions arise after the polarity of epithelial cells is established which sets the foundation for further development and specification. Within the blastula, inner blastomeres are generally non-polar while epithelial cells demonstrate polarity. [ 16 ]


Mammalian embryos undergo compaction around the 8-cell stage where  E-cadherins  as well as  alpha  and  beta   catenins  are expressed. This process makes a ball of embryonic cells which are capable of interacting, rather than a group of diffuse and undifferentiated cells. E-cadherin adhesion defines the  apico-basal axis  in the developing embryo and turns the embryo from an indistinct ball of cells to a more polarized  phenotype  which sets the stage for further development into a fully formed blastocyst. [ 16 ]


Xenopus  membrane polarity is established with the first cell cleavage. Amphibian EP-cadherin and XB/U cadherin perform a similar role as E-cadherin in mammals establishing blastomere polarity and solidifying cell-cell interactions which are crucial for further development. [ 16 ]


Experiments with implantation in mice show that  hormonal induction ,  superovulation  and  artificial insemination  successfully produce preimplantation mouse embryos. In the mice, ninety percent of the females were induced by mechanical stimulation to undergo pregnancy and implant at least one embryo. [ 17 ]  These results prove to be encouraging because they provide a basis for potential implantation in other mammalian species, such as humans.


Blastula-stage cells can behave as pluripotent stem cells in many species. Pluripotent stem cells are the starting point to produce organ specific cells that can potentially aid in repair and prevention of injury and degeneration. Combining the expression of  transcription factors  and locational positioning of the blastula cells can lead to the development of induced functional organs and tissues. Pluripotent  Xenopus  cells, when used in an in vivo strategy, were able to form into functional  retinas . By transplanting them to the eye field on the  neural plate , and by inducing several mis-expressions of transcription factors, the cells were committed to the retinal lineage and could guide vision based behavior in the  Xenopus . [ 18 ]


Keith Henry Stockman Campbell  (23 May 1954 – 5 October 2012) [ 1 ]  was a British  biologist  who was a member of the team at  Roslin Institute  that in 1996 first cloned a mammal, a  Finnish Dorset  lamb named  Dolly , from fully differentiated adult mammary cells. He was Professor of Animal Development at the  University of Nottingham . [ 2 ] [ 3 ] [ 4 ] [ 5 ] [ 6 ] [ 7 ]  In 2008, he received the  Shaw Prize  for Medicine and Life Sciences jointly with  Ian Wilmut  and  Shinya Yamanaka  for "their works on the cell differentiation in mammals".


Campbell was born in  Birmingham , England, to an English mother and Scottish father. He started his education in  Perth , Scotland, but, when he was eight years old, his family returned to Birmingham, where he attended  King Edward VI Camp Hill School for Boys . [ 8 ]  He obtained his Bachelor of Science degree in  microbiology  from the  Queen Elizabeth College ,  University of London  (now part of  King's College London ). [ 9 ]  In 1983 Campbell was awarded the  Marie Curie  Research Scholarship, which led to postgraduate studies and later his PhD from the  University of Sussex [ 10 ]  (Brighton, England, UK). [ 11 ] [ 12 ] [ 13 ]


Campbell's interest in cloning mammals was inspired by work done by Karl Illmensee and  John Gurdon . [ citation needed ]  Working at the  Roslin Institute  since 1991, Campbell became involved with the cloning efforts led by  Ian Wilmut . In July 1995 Keith Campbell and Bill Ritchie succeeded in producing a pair of lambs, Megan and Morag from embryonic cells, which had differentiated in culture.


In 1996, a team led by  Ian Wilmut  with Keith Campbell as the main contributor, used the same technique and shocked the world by successfully cloning a sheep from adult mammary cells. Dolly, a Finn Dorset sheep named after the singer  Dolly Parton , was born in 1996 and lived to be six years old (dying from a viral infection and not old age, as has been suggested). Campbell had a key role in the creation of Dolly, as he had the crucial idea of co-ordinating the stages of the "cell cycle" of the donor somatic cells and the recipient eggs and using diploid quiescent or "G0" arrested somatic cells as nuclear donors. In 2006,  Ian Wilmut  admitted that Campbell deserved "66 per cent" of the credit. [ 14 ]


In 1997, Ritchie and Campbell in collaboration with PPL (Pharmaceutical Proteins Limited) created another sheep named "Polly", created from genetically altered skin cells containing a human gene. [ 15 ]  In 2000, after joining PPL Ltd, Campbell and his PPL team (based in North America) were successful in producing the world's first piglets by  Somatic-cell nuclear transfer  (SCNT), the so-called cloning technique. Furthermore, the PPL teams based in Roslin, Scotland and Blacksburg (USA) used the technique to produce the first gene targeted domestic animals as well as a range of animals producing human therapeutic proteins in their milk. [ 16 ] [ 17 ]


From November 1999, Campbell held the post of Professor of Animal Development, Division of Animal Physiology, School of Biosciences at the  University of Nottingham  where he continued to study embryo growth and differentiation. He supported the use of SCNT for the production of personalised stem cell therapies and for the study of human diseases and the use of cybrid embryo production to overcome the lack of human eggs available for research. Stem cells can be isolated from embryonic, fetal and adult derived material and more recently by overexpression of certain genes for the production of "induced pluripotent cells". Campbell believed all potential stem cell populations should be used for both basic and applied research which may provide basic scientific knowledge and lead to the development of cell therapies. [ 18 ] [ 19 ] [ 20 ] [ 21 ]


In 2008, he received the  Shaw Prize  for Medicine and Life Sciences jointly with Ian Wilmut and Shinya Yamanaka. [ 22 ]  He was awarded the Pioneer Award from the International Embryo Transfer Society posthumously in 2015. [ 23 ]


Campbell died on 5 October 2012, aged 58, after accidentally hanging himself in his bedroom at his  Ingleby, Derbyshire  home, whilst heavily intoxicated. It was determined at the inquest that he had been behaving erratically at the time and had no actual intention to kill himself; the verdict was a  death by misadventure . [ 24 ] [ 25 ]  He was buried at Bretby Crematorium,  Derbyshire . [ 26 ]  He is survived by his wife, Kathy, and two daughters, Claire and Lauren.


Christians take multiple positions in the debate on the  morality of human cloning . Since  Dolly  the sheep was successfully cloned on 5 July 1996, and the possibility of cloning humans became a reality, Christian leaders have been pressed to take an ethical stance on its  morality . While many Christians tend to disagree with the practice, such as  Roman Catholics  and a majority of  fundamentalist  pastors, including  Southern Baptists , the views taken by various other  Christian denominations  are diverse and often conflicting. [ 1 ]  It is hard to pinpoint any one, definite stance of the Christian religion, since there are so many  Christian denominations  and so few official statements from each of them concerning the morality of human cloning.


There are certain  Protestant denominations  that do not disagree with the acceptability of  human cloning . Mary Seller, for example, a member of the Church of England's Board of Social Responsibility and a professor of developmental genetics, states, "Cloning, like all science, must be used responsibly. Cloning humans is not desirable. But cloning sheep has its uses." [ 1 ]  On the other hand, according to a survey of Christian fundamentalist pastors, responses indicated a "common account of human cloning as primarily reproductive in nature, proscribed by its violation of God's will and role." [ 1 ]  Many of these pastors acknowledged the reason for this violation being rooted in the religiously motivated view that human cloning is an example of scientists 'playing God.'" [ 1 ]  It is not only this that many Christians are concerned about, however; other concerns include whether the dignity of the human person is overlooked, as well as the role of the parents as co-creators. All of these things may contribute to why many fundamentalist Christian pastors see human reproductive cloning as simply "forbidden territory." [ 1 ]


Some scientists do argue that the plurality of views comes from the differing understandings of what exactly  human cloning  is. In Weasel and Jensen's survey on "Language and Values," they found that Christian pastors used a "consistent emphasis on  DNA ." [ 1 ]  Scientists describing the same processes used terminology such as, "insert an isolated  nucleus  from the donor to produce a dividing and viable  embryo " into an enucleated egg" [ 1 ]  One reason why Christian  fundamentalist  pastors would be more apt to use terminology which includes "DNA" more prominently is that DNA is a "value-laden" term which carries religious significance. [ 1 ]  Therefore, not only is DNA often equated, in religious circles, with one's genetic identity, but also with one's singular, "God-given" identity.


Another point of misunderstanding arises in the separation of human  reproductive cloning  and  therapeutic cloning . While most Christians do not support reproductive cloning, therapeutic cloning is a branch of cloning which has garnered slightly more support from certain denominations. Some Christian traditions, such as the  United Church of Christ , believe that cloning can be conducted ethically for this purpose, in order to help treat disease and improve the public health, as they do not see the fertilized  ovum  as constituting a person. Courtney Campbell, director of the Program for Ethics, Science and the Environment at  Oregon State University , says, "Some traditions and leading figures in conservative Protestantism who were opposed to human cloning for reproductive reasons have come to see that given the ambiguity about their own views about the status of embryonic life, and given the potential for health benefits, they could be opposed to reproductive cloning, but affirm therapeutic cloning. [ 2 ]  The main reason, she says, is the tradition of emphasizing the individual choice over central dogma.


Catholic support for therapeutic cloning is conditional on the stipulation that it does not involve any kind of infringement upon human embryos.  John Paul II  states, "These techniques, insofar as they involve the manipulation and destruction of human embryos, are not morally acceptable, even when their proposed goal is good in itself." [ 3 ]  Thus, according to the Catholic ethical system, even a good that might come from therapeutic cloning would be morally unacceptable, as it would be the result of an act which, according to Catholics, is itself intrinsically immoral. This, for many Christians, is hard to abide by, for, as one Professor of Christian Ethics states, "Our society is preoccupied with death and the pursuit of health seems to justify anything." [ 4 ]


The  Church of England  put out a statement on the Church's website which reads, "human reproductive cloning was made unlawful by the  Human Fertilisation and Embryology Act 1990 . Few members of the Church of England would dissent from such a position. However, therapeutic cloning may be thought of as ethical, as it does not result in another human being." [ 5 ]  Thus, while reproductive cloning is again discouraged, therapeutic cloning is more acceptable.


In a statement given by the  Lutheran Church–Missouri Synod , on the other hand, therapeutic cloning was rejected as "contrary to the Word of God and our 1998 resolution". [ 6 ]  For this particular group, therapeutic cloning is assumed to involve the destruction of human embryos, and is therefore against the synod's resolution to "reaffirm and celebrate life" and "protect the sanctity of human life". [ 6 ]


Christian organizations are challenged to respond to a number of concerns about the issue of the  dignity  of the human person, especially in relation to scientific study. Much of the debate has to do with the question of at what point the  soul  enters the body. Catholics believe that the soul enters the body at the moment of conception when the  sperm  and  egg  unite. [ 7 ]  Thus, Catholics and other Christian denominations that share this belief may see embryonic cloning as tantamount to live human experimentation and therefore contrary to God's will.


Most Christians believe that a person has intrinsic  dignity  based on his being created in the image and likeness of  God  and in his call to communion with God. [ 8 ]


Many Christians that do not agree on an exact definition of  dignity , which leads some scientists to ignore this concern, seeing it as a vague excuse that some Christians use to justify cloning bans [ 9 ]  Or, if a definition is agreed upon, it may be challenged as being too weak, so that, according to Steven Pinker, a Professor of Psychology at Harvard University, it is "hardly up to the heavyweight moral demands assigned to it". [ 10 ]


The belief in intrinsic  dignity , nonetheless, leads the Christians who hold this belief to also argue that if the  soul  enters the body at the moment when the  sperm  and the  egg  unite, producing cloned  zygotes  that are unlikely to survive is equivalent to murder. Therefore, if one believes, as Catholics do, that the zygotes have souls and are therefore human, in the words of  John Paul II , "regardless of the objective for which it was done, human embryonic cloning conflicts with the international legal norms that protect human dignity." [ 3 ]  Some Christian conservatives even express concern that cloned embryos would have no soul, since it is, in their view, born outside of God's parameters, [ 11 ]  as its creation is in a laboratory setting rather than natural conception.


Certain Christians also hold that, in the act of creating a human being, his or her dignity is also upheld by the presence of a loving union between the two partners, so that "only when love exists should the creation of other persons be envisaged." [ 12 ]  Because of this, "divine creation and co-creation are similar". [ 12 ]  In the act of scientific  cloning , however, the  embryo  is a result of the artificial union of the  sperm  and the  egg , so that "the presence of love between the co-creators does not exist." [ 12 ]  Thus, according to this view, the dignity of the person is not upheld. Rev. Demetri Demopulos,  Greek Orthodox   pastor  and  geneticist , states, "As an  Orthodox Christian , I speak out in opposition to any attempt to clone a human being because humans are supposed to be created by acts of love between two people, not through the manipulation of cells in acts that are ultimately about self-love."


Much of the concern related to human cloning stems from a fear of overstepping the bounds of the natural order. Through the act of creating another human being, Christians have also posed the question as to whether this would be too much like playing God. [ 13 ]  Not only this, but some Christian  ethicists  argue that human cloning would "create substantial issues of identity and individuality". [ 4 ]  This could be seen through the confusion of the child resulting from cloning as to who his parents are, what connection he has to his genetic duplicate, etc.


Despite the ethical gray area surrounding the act of cloning itself, most, if not all Christians, still hold that children who may result from the process should be loved and cared for as much as any other child, since they would be considered fully human [ 12 ]  and therefore reflect the  Divine  image, as defined by  Gaudium et spes , a document of the Second Vatican Council. [ 8 ]  However, according to Richard McCormick,  S.J. , cloning disrupts the familial order. It "would involve removing  insemination  and  fertilization  from the marriage relationship, and it would also remove one of the partners from the entire process". According to Stephen G. Post, a Catholic journalist, "[removal] of the male impregnators from the procreative  dyad  would simply drive the nail into the coffin of fatherhood, unless one thinks that biological and social fatherhood are utterly disconnected". [ 14 ]


Rev. Demetri Demopulos,  Greek Orthodox  pastor and  geneticist :


Richard McCormick, a  Jesuit  priest and professor of Christian  ethics  at the  University of Notre Dame :


Mary Seller, a member of  Church of England 's Board of Social Responsibility and a professor of developmental genetics:


Pope John Paul II :


United Church of Christ  Committee on  Genetics :


Ben Mitchellin, assistant professor of Christian  ethics  at  Southern Baptist  Theological Seminary:


A  clone  is a group of  identical  cells that share a  common ancestry , meaning they are derived from the same cell. [ 1 ]


Clonality implies the state of a cell or a substance being derived from one source or the other. Thus there are terms like  polyclonal —derived from  many  clones;  oligoclonal [ 2 ] —derived from a  few  clones; and  monoclonal —derived from  one  clone. These terms are most commonly used in context of  antibodies  or  immunocytes .


This concept of clone assumes importance as all the cells that form a clone share common ancestry, which has a very significant consequence: shared  genotype .


Most other cells cannot divide indefinitely as after a few cycles of cell division the cells stop expressing an enzyme  telomerase . The genetic material, in the form of  deoxyribonucleic acid (DNA) , continues to shorten with each cell division, and cells eventually stop dividing when they sense that their DNA is critically shortened. However, this enzyme in "youthful" cells replaces these lost bits ( nucleotides ) of DNA, thus making almost unlimited cycles of cell division possible. It is believed that the above-mentioned tissues have a constitutional elevated expression of telomerase. When ultimately many cells are produced by a single cell,  clonal expansion  is said to have taken place.


A somewhat similar concept is that of a  clonal colony  (also called a  genet ), wherein the cells (usually unicellular) also share a common ancestry, but which also requires the products of clonal expansion to reside at "one place", or in close proximity. A clonal colony would be well exemplified by a  bacterial culture  colony, or the bacterial films that are more likely to be found  in vivo  (e.g., in infected multicellular hosts). Whereas, the cells of clones dealt with here are specialized cells of a multicellular organism (usually vertebrates), and reside at quite distant places. For instance, two plasma cells belonging to the same clone could be derived from different memory cells (in turn with shared clonality) and could be residing in quite distant locations, such as the cervical (in the neck) and inguinal (in the groin)  lymph nodes .


The single-cell  eukaryote   Paramecium  tetraurelia  can undergo both asexual and  sexual reproduction .  Asexual or clonal reproduction occurs by  binary fission .  Binary fission involves  mitosis -like behavior of the chromosomes similar to that of cells in higher organisms.  The sexual forms of reproduction are  autogamy , a kind of self-fertilization, and  conjugation , a kind of sexual interation between different cells.  Clonal asexual reproduction can be initiated after completion of autogamy or conjugation.   P. tetraurelia  is able to replicate asexually for many generations but the dividing cells gradually age and after about 200 cell divisions, if the cells fail to undergo another autogamy or conjugation, they lose vitality and expire.  This process is referred to as clonal aging.  Experiments by Smith-Sonneborn, [ 7 ]  Holmes and Holmes [ 8 ]  and Gilley and Blackburn [ 9 ]  showed that accumulation of  DNA damage  is the likely cause of clonal aging in  P. tetraurelia . This aging process has similarities to the aging process in multicellular eukaryotes (See  DNA damage theory of aging ).


Clone Manager  is a commercial  bioinformatics  software work suite of Sci-Ed, that supports  molecular biologists  with data management and allows them to perform certain  in silico  preanalysis. [ 1 ]


This type of bioinformatics software is used for managing, analyzing and visualizing DNA and protein sequence data essential for molecular biology. [ 2 ]


For enzyme read control, sequence processing of identical individuals, cloning simulation, graphic map drawing, primer design and analysis, global and local sequence alignment, similarity search, laboratory scale sequence assembly projects A comprehensive set of tools.


This  scientific software  article is a  stub . You can help Wikipedia by  expanding it .


A  cloning vector  is a small piece of  DNA  that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for  cloning  purposes. [ 1 ]  The cloning vector may be DNA taken from a  virus , the  cell  of a higher organism, or it may be the  plasmid  of a bacterium. The  vector  contains features that allow for the convenient insertion of a DNA fragment into the vector or its removal from the vector, for example through the presence of  restriction sites . The vector and the foreign DNA may be treated with a  restriction enzyme  that cuts the DNA, and DNA fragments thus generated contain either blunt ends or overhangs known as sticky ends, and vector DNA and foreign DNA with compatible ends can then be joined by  molecular ligation .  After a DNA fragment has been cloned into a cloning vector, it may be further  subcloned  into another vector designed for more specific use.


There are many types of cloning vectors, but the most commonly used ones are genetically engineered  plasmids .  Cloning is generally first performed using  Escherichia coli ,  and cloning vectors in  E. coli  include plasmids,  bacteriophages  (such as  phage λ ),  cosmids , and  bacterial artificial chromosomes  (BACs).  Some DNA, however, cannot be stably maintained in  E. coli , for example very large DNA fragments, and other organisms such as yeast may be used.  Cloning vectors in yeast include  yeast artificial chromosomes  (YACs).


All commonly used cloning vectors in  molecular biology  have key features necessary for their function, such as a suitable cloning site and selectable marker.  Others may have additional features specific to their use.  For reason of ease and convenience, cloning is often performed using  E. coli . Thus, the cloning vectors used often have elements necessary for their propagation and maintenance in  E. coli , such as a functional  origin of replication  (ori).  The  ColE1  origin of replication is found in many plasmids.  Some vectors also include elements that allow them to be maintained in another organism in addition to  E. coli , and these vectors are called  shuttle vector .


All cloning vectors have features that allow a gene to be conveniently inserted into the vector or removed from it. This may be a  multiple cloning site  (MCS) or polylinker, which contains many unique  restriction sites .  The restriction sites in the MCS are first cleaved by restriction enzymes, then a  PCR -amplified target gene also digested with the same enzymes is ligated into the vectors using  DNA ligase . The target DNA sequence can be inserted into the vector in a specific direction if so desired.  The restriction sites may be further used for  sub-cloning  into another vector if necessary. [ citation needed ]


Other cloning vectors may use  topoisomerase  instead of ligase and cloning may be done more rapidly without the need for restriction digest of the vector or insert.  In this  TOPO cloning  method a linearized vector is activated by attaching topoisomerase I to its ends, and this "TOPO-activated" vector may then accept a PCR product by ligating both the 5' ends of the PCR product, releasing the topoisomerase and forming a circular vector in the process. [ 2 ]   Another method of cloning without the use of DNA digest and ligase is by  DNA recombination , for example as used in the  Gateway cloning system . [ 3 ] [ 4 ]  The gene, once cloned into the cloning vector (called entry clone in this method), may be conveniently introduced into a variety of expression vectors by recombination. [ 5 ]


A  selectable marker  is carried by the vector to allow the selection of positively  transformed  cells.   Antibiotic  resistance is often used as marker, an example being the  beta-lactamase  gene, which confers resistance to  the  penicillin  group of  beta-lactam antibiotics  like  ampicillin .  Some vectors contain two selectable markers, for example the plasmid pACYC177 has both ampicillin and  kanamycin  resistance gene. [ 6 ]  Shuttle vector which is designed to be maintained in two different organisms may also require two selectable markers, although some selectable markers such as resistance to  zeocin  and  hygromycin B  are effective in different cell types.   Auxotrophic  selection markers that allow an auxotrophic organism to grow in  minimal growth medium  may also be used; examples of these are  LEU2  and  URA3  which are used with their corresponding auxotrophic strains of yeast. [ 7 ]


Another kind of selectable marker allows for the positive selection of plasmid with cloned gene.  This may involve the use of a gene lethal to the host cells, such as  barnase , [ 8 ]   Ccda , [ 9 ]  and the  parD/parE  toxins. [ 10 ] [ 11 ]   This typically works by disrupting or removing the lethal gene during the cloning process, and unsuccessful clones where the lethal gene still remains intact would kill the host cells, therefore only successful clones are selected.


Reporter genes are used in some cloning vectors to facilitate the screening of successful clones by using features of these genes that allow successful clone to be easily identified.  Such features present in cloning vectors may be the  lacZ α fragment  for α complementation in  blue-white selection , and/or  marker gene  or  reporter genes  in frame with and flanking the  MCS  to facilitate the production of  fusion proteins .  Examples of fusion partners that may be used for screening are the  green fluorescent protein  (GFP) and  luciferase .


A cloning vector need not contain suitable elements for the  expression  of a cloned target gene, such as a  promoter  and  ribosomal binding site  (RBS),  many however do, and may then work as an  expression vector .  The target  DNA  may be inserted into a site that is under the control of a particular promoter necessary for the expression of the target gene in the chosen host.   Where the promoter is present, the expression of the gene is preferably tightly controlled and  inducible  so that proteins are only produced when required.  Some commonly used promoters are the  T7  and  lac  promoters .  The presence of a promoter is necessary when screening techniques such as  blue-white selection  are used.


Cloning vectors without promoter and RBS for the cloned DNA sequence are sometimes used, for example when cloning genes whose products are toxic to  E. coli  cells.  Promoter and RBS for the cloned DNA sequence are also unnecessary when first making a  genomic  or  cDNA library  of clones since the cloned genes are normally subcloned into a more appropriate expression vector if their expression is required.


Some vectors are designed for transcription only with no heterologous protein expressed, for example for  in vitro  mRNA production.  These vectors are called transcription vectors.  They may lack the sequences necessary for polyadenylation and termination, therefore may not be used for protein production.


A large number of cloning vectors are available, and choosing the vector may depend upon a number of factors, such as the size of the insert, copy number and cloning method.  Large insert may not be stably maintained in a general cloning vector, especially for those with a high copy number, therefore cloning large fragments may require more specialised cloning vector. [ 6 ]


Plasmids are autonomously replicating circular extra-chromosomal DNA. They are the standard cloning vectors and the ones most commonly used.  Most general plasmids may be used to clone DNA inserts of up to 15 kb in size.  One of the earliest commonly used cloning vectors is the  pBR322  plasmid.  Other cloning vectors include the  pUC  series of plasmids, and a large number of different cloning plasmid vectors are available.  Many plasmids have high copy numbers, for example,  pUC19  has a copy number of 500-700 copies per cell, [ 6 ]  and high copy number is useful as it produces greater yield of recombinant plasmid for subsequent manipulation. However low-copy-number plasmids may be preferably used in certain circumstances, for example, when the protein from the cloned gene is toxic to the cells. [ 12 ]


Some plasmids contain an  M13 bacteriophage  origin of replication and may be used to generate single-stranded DNA.  These are called  phagemids , and examples are the  pBluescript  series of cloning vectors.


The bacteriophages used for cloning are the  λ phage  and  M13 phage . [ 13 ]   There is an upper limit on the amount of DNA that can be packed into a phage (a maximum of 53 kb), therefore to allow foreign DNA to be inserted into phage DNA, phage cloning vectors may need to have some non-essential genes deleted, for example the genes for  lysogeny  since using phage λ as a cloning vector involves only the lytic cycle. [ 14 ]   There are two kinds of λ phage vectors - insertion vector and replacement vector.  Insertion vectors contain a unique cleavage site whereby foreign DNA with size of 5–11 kb may be inserted.   In replacement vectors, the cleavage sites flank a region containing genes not essential for the lytic cycle, and this region may be deleted and replaced by the DNA insert in the cloning process, and a larger sized DNA of 8–24 kb may be inserted. [ 6 ]


There is also a lower size limit for DNA that can be packed into a phage, and vector DNA that is too small cannot be properly packaged into the phage.  This property can be used for selection - vector without insert may be too small, therefore only vectors with insert may be selected for propagation. [ 15 ]


Cosmids  are plasmids that incorporate a segment of bacteriophage λ DNA that has the cohesive end site ( cos ) which contains elements required for packaging DNA into λ particles. Under apt origin of replication (ori), it can replicate as a plasmid.  It is normally used to clone large DNA fragments between 28 and 45 Kb. [ 6 ]


Insert size of up to 350 kb can be cloned in  bacterial artificial chromosome  (BAC). BACs are maintained in  E. coli  with a copy number of only 1 per cell. [ 6 ]   BACs are based on  F plasmid , another artificial chromosome called the  PAC  is based on the  P1 phage .


Yeast artificial chromosome  are used as vectors to clone DNA fragments of more than 1 mega base (1Mb=1000kb) in size. They are useful in cloning larger DNA fragments as required in mapping genomes such as in the  Human Genome Project . It contains a telomeric sequence, an autonomously replicating sequence (features required to replicate linear chromosomes in yeast cells). These vectors also contain suitable restriction sites to clone foreign DNA as well as genes to be used as selectable markers.