Index
It is the process of producing genetically identical individuals of an organism, either naturally or artificially.
In nature, many organisms produce clones through asexual reproduction.
Cloning in biotechnology refers to creating clones of organisms or copies of cells or fragments of DNA (molecular cloning). Beyond biology, the term refers to producing multiple digital media or software copies.
The term clone, invented by JBS Haldane, is derived from the ancient Greek word κλών klōn, “twig,” which refers to the process by which a new plant can be created from a twig. In botany, the term lusus was traditionally used.
In horticulture, the spelling clone was used until the 20th century; the ending e came into use to indicate that the vowel is a long “o” rather than a short “o.” The spelling clone has been used exclusively since the term entered the popular lexicon more broadly.
Natural cloning
Cloning is a natural form of reproduction that has allowed life forms to spread for hundreds of millions of years.
It is the method of reproduction used by plants, fungi, and bacteria, and it is also how to clone colonies reproduce.
These organisms include blueberry plants, hazelnuts, Pando trees, the Kentucky coffeetree, Myrica, and the American sweetgum.
Molecular cloning
Molecular cloning is a set of experimental molecular biology methods that assemble recombinant DNA molecules and direct their replication within host organisms.
The use of cloning refers to replicating a molecule to produce a population of cells with identical DNA molecules.
Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned and the species that will serve as a living host for recombinant DNA replication.
Molecular cloning methods are central to many contemporary areas of modern biology and medicine.
In a conventional molecular cloning experiment, the DNA to be cloned is obtained from an organism of interest, then treated with enzymes in the test tube to generate smaller DNA fragments.
These fragments are then combined with vector DNA to generate recombinant DNA molecules. The recombinant DNA is then introduced into a host organism (typically an easy-to-grow benign laboratory strain of E. coli bacteria).
This will generate a population of organisms in which the recombinant DNA molecules will replicate along with the host’s DNA. Because they contain foreign DNA fragments, these are transgenic or genetically modified (GMO) microorganisms.
This process takes advantage of the fact that a single bacterial cell can be induced to take up and replicate a single recombinant DNA molecule.
This individual cell can expand exponentially to generate large numbers of bacteria, each containing copies of the original recombinant molecule. Therefore, both the resulting bacterial population and the recombinant DNA molecule are commonly referred to as “clones.”
Strictly speaking, recombinant DNA refers to DNA molecules, while molecular cloning refers to the experimental methods used to assemble them.
The idea arose that different DNA sequences could be inserted into a plasmid and that these foreign sequences would be transported to bacteria and digested as part of the plasmid. That is, these plasmids could serve as cloning vectors to carry genes.
Virtually any DNA sequence can be cloned and amplified, but some factors can limit the success of the process. DNA sequences that are difficult to clone are inverted repeats, replication origins, centromeres, and telomeres.
Another feature that limits the chances of success is the large size of the DNA sequence. Inserts larger than ten kbp have minimal success, but bacteriophages such as bacteriophage λ can be modified to insert a series of up to 40 kbp successfully.
Cell cloning
Cloning of single-celled organisms
Cloning a cell means deriving a population of cells from a single cell. In the case of single-celled organisms such as bacteria and yeast, this process is straightforward and essentially only requires the injection of the appropriate medium.
However, cell cloning is an arduous task in cell cultures of multicellular organisms as these cells will not increase in traditional media.
A proper tissue culture technique used to clone different lineages of cell lines involves cloning rings (cylinders).
In this technique, a single cell suspension of cells that have been exposed to a mutagenic agent or drug used to direct selection is plated at high dilution to create isolated colonies, each of which comes from a single cell and potentially distinct clonal.
In an early growth stage, when colonies consist of only a few cells, sterile polystyrene rings (cloning rings), dipped in fat, are placed on an individual settlement, and a small amount of trypsin is added.
Cloned cells are harvested inside the ring and transferred to a new vessel for further growth.
Stem cell cloning
Somatic cell nuclear transfer, known as SCNT for short, can also be used to create embryos for research or therapeutic purposes.
The most likely purpose is to produce embryos for stem cell research. This process is also called “research cloning” or “therapeutic cloning.”
The goal is not to create cloned humans (called “reproductive cloning”) but rather to collect stem cells that can be used to study human development and potentially treat disease.
While a clonal human blastocyst has been created, the stem cell lines have not yet been isolated from a clonal source.
Therapeutic cloning is accomplished by creating embryonic stem cells to treat diseases such as diabetes and Alzheimer’s. The process begins by removing the nucleus (which contains DNA) from an egg and inserting a seat from the adult cell to be cloned.
In the case of someone with Alzheimer’s disease, the nucleus of a skin cell from that patient is placed in an empty egg. The reprogrammed cell develops into an embryo because the egg reacts with the transferred heart.
The embryo will become genetically identical to the patient. The source will form a blastocyst that has the potential to create/develop into any cell in the body.
Somatic cell nuclear transfer is used for cloning because somatic cells can be easily acquired and grown in the laboratory. This process can add or remove specific genomes of farm animals.
The critical point is that cloning is accomplished when the oocyte maintains normal functions. Instead of using the sperm and genomes of the egg to replicate, the oocyte inserts itself into the nucleus of the donor somatic cell.
The oocyte will react in the nucleus of the somatic cell in the same way as it would in sperm cells.
Cloning a particular farm animal using somatic cell nuclear transfer is relatively the same for all animals. The first step is to collect the somatic cells from the animal to be cloned.
Somatic cells can be used immediately or stored in the laboratory for later use. The most challenging part of somatic cell nuclear transfer is the removal of maternal DNA from an oocyte at metaphase II.
Once this is done, the somatic nucleus can be inserted into an egg cytoplasm. This creates a one-cell embryo. The grouped somatic cell and egg cytoplasm are introduced into an electrical current.
This energy is expected to allow the cloned embryo to begin development. Successfully developed seeds are then placed in surrogate recipients, such as a cow or sheep, in the case of farm animals.
Somatic cell nuclear transfer is considered an excellent method to produce agricultural animals for food consumption. Sheep, cows, goats, and pigs were successfully cloned.
Another benefit is somatic cell nuclear transfer, which is a solution for cloning endangered species on the verge of extinction.
However, the stresses placed on both the ovum and the introduced nucleus can be enormous, leading to a significant loss of cells and early investigations.
For example, the cloned sheep Dolly was born after 277 eggs were used for somatic cell nuclear transfer, which created 29 viable embryos. Only three of these embryos survived birth, and only one survived to adulthood.
As the procedure could not be automated and performed manually under the microscope, somatic cell nuclear transfer was resource-intensive.
The biochemistry involved in reprogramming the differentiated somatic cell nucleus and activating the receptor ovum was not well understood.
However, in 2014 researchers reported cloning success rates of seven to eight out of ten, and in 2016, a Korean company Sooam Biotech was said to be producing 500 cloned embryos per day.
In the somatic cell, nuclear transfer, not all genetic information from the donor cell is transferred, as the donor cell’s mitochondria containing its own mitochondrial DNA are left behind.
The resulting hybrid cells retain the mitochondrial structures that initially belonged to the egg. Consequently, clones like Dolly born from somatic cell nuclear transfer are not perfect copies of the donor nucleus.
Cloning of organisms
Organism cloning (also called reproductive cloning) creates a new multicellular organism genetically identical to another.
This form of cloning is an asexual reproduction method, where fertilization or contact between gametes does not occur.
Asexual reproduction is a natural phenomenon in many species, including most plants and some insects. Scientists have made some crucial achievements with cloning, including the asexual reproduction of sheep and cows.
There is a great ethical debate about whether to use cloning or not. However, asexual cloning or propagation has been standard in the horticultural world for hundreds of years.
Horticultural
The term clone is used in horticulture to refer to the offspring of a single plant produced by vegetative reproduction or apomixis.
Many horticultural plant cultivars are clones derived from a single individual, multiplied by some process other than sexual reproduction.
For example, some European grape cultivars represent clones that have been propagated for more than two millennia. Other examples are potatoes and bananas.
Grafting can be regarded as cloning, as all buds and branches from the graft are genetically clones of a single individual. Still, this type of cloning has not been the subject of ethical scrutiny and is generally treated as a completely different type of operation.
Many trees, shrubs, vines, ferns, and other herbaceous perennials form clonal colonies. The parts of an individual plant can be separated by fragmentation and grow to become separate clonal individuals.
A typical example is the vegetative reproduction of clones of moss and agrimony gametophytes using gemmae.
Some vascular plants, such as dandelions and certain viviparous herbs, also form seeds asexually, called apomixis, resulting in clonal populations of genetically identical individuals.
Parthenogenesis
Clonal derivation exists in nature in some animal species and is known as parthenogenesis (reproduction of an organism by itself without a partner).
This form of asexual reproduction is only found in the females of some insects, crustaceans, nematodes, fish (for example, the hammerhead shark), the Komodo dragon, and lizards.
Growth and development occur without fertilization by a man. In plants, parthenogenesis means the result of an embryo from an unfertilized egg and is a component process of apomixis.
The offspring will always be female in species that use the XY sex-determination system. An example is the small fire ant (Wasmannia auropunctata), native to Central and South America but has spread through many tropical environments.
Artificial cloning of organisms
Artificial cloning of organisms can also be called reproductive cloning.
First steps
Hans Spemann, a German embryologist, received the Nobel Prize in Physiology or Medicine in 1935 for his discovery of the effect now known as embryonic induction, exerted by various parts of the embryo, which directs the development of groups of cells in particular tissues and organs.
In 1928, he and his student, Hilde Mangold, were the first to perform somatic cell nuclear transfer using amphibian embryos, one of the first steps toward cloning.
Methods
Reproductive cloning generally uses somatic cell nuclear transfer to create genetically identical animals.
This process involves the transfer of a nucleus from a donor adult cell (somatic cell) to an egg from which the heart has been removed or a cell from a blastocyst from which the seat has been removed.
If the egg begins to divide normally, it is transferred to the surrogate’s uterus. Such clones are not identical since somatic cells can contain mutations in their nuclear DNA.
In addition, the mitochondria in the cytoplasm also contain DNA, and during somatic cell nuclear transfer, this mitochondrial DNA is entirely from the cytoplasmic donor egg. Therefore, the mitochondrial genome is not the same as that of the donor cell from the nucleus.
This may have important implications for interspecies nuclear transfer in which nuclear mitochondrial incompatibilities can lead to death.
Artificial embryo division or embryo twinning, a technique that creates monozygotic twins from a single embryo, is not viewed the same way as other cloning methods.
During that procedure, a donor embryo is divided into two separate sources, which can be transferred through embryo transfer.
It is optimally performed at the 6 to 8-cell stage, where it can be used as an expansion of in vitro fertilization (IVF) to increase the number of available embryos. If both sources are successful, it gives rise to monozygotic (identical) twins.
Dolly the sheep
Dolly, a Finn-Dorset sheep, was the first mammal to be successfully cloned from an adult somatic cell. Dolly was formed by taking a cell from the udder of her 6-year-old biological mother.
Dolly’s embryo was created by taking the cell and inserting it into a sheep egg. It took 434 tries before an origin was successful. The source was placed inside a female sheep who had a normal pregnancy.
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 she died in 2003 when she was six years old.
He was born on July 5, 1996, but was not announced until February 22, 1997. His stuffed remains were placed in the Royal Museum in Edinburgh, part of the National Museums of Scotland.
Dolly was publicly significant because the effort demonstrated that the genetic material of a specific adult cell, programmed to express only a distinct subset of its genes, can be reprogrammed to grow an entirely new organism.
Before this demonstration, John Gurdon had shown that the nuclei of differentiated cells could give rise to a complete organism after transplantation into an enucleated egg. However, this concept has yet to be proven in a mammalian system.
The first mammalian cloning (resulting in Dolly, the sheep) 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 young. The first successfully cloned horse, Promethea, made 814 attempts.
In particular, although the first clones were frogs, no adult cloned frog has yet been produced from an adult somatic nucleus donor cell.
Early claims were that Dolly the sheep had pathologies that resembled accelerated aging.
Scientists speculated that Dolly’s death in 2003 was related to shortening 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 premature death due to respiratory infection was not related to shortcomings in the cloning process.
This idea that nuclei have not irreversibly aged was shown in 2013 to be true for mice.
Dolly was named after actress Dolly Parton because the cells cloned to make her were from a mammary gland, and Parton is known for her wide cleavage.
Human colonization
Human cloning is creating a genetically identical copy of a human being. The term is generally used to refer to artificial human cloning, which reproduces human cells and tissues.
It does not refer to the natural conception and delivery of identical twins. The possibility of human cloning has generated controversy. These ethical concerns have led several nations to pass the legislature regarding human cloning and its legality.
The scientists have no intention of attempting to clone people and believe their results should spark a broader discussion about the laws and regulations the world needs to regulate cloning.
Two commonly discussed types of theoretical human cloning are therapeutic cloning and reproductive cloning.
Therapeutic cloning involves cloning cells from a human for use in medicine and transplantation and is an active area of research, but not in medical practice anywhere in the world as of 2014.
Two standard therapeutic cloning methods being investigated are somatic cell nuclear transfer and, more recently, pluripotent stem cell induction.
Reproductive cloning would involve making a cloned entire human, rather than just specific cells or tissues.
Ethical issues
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 also faced by secular perspectives.
The perspectives on human cloning are theoretical, as reproductive and therapeutic human cloning is not used commercially; animals are currently cloned in laboratories and livestock production.
Advocates support the development of therapeutic cloning to generate entire tissues and organs to treat patients who are otherwise unable to obtain transplants, avoid the need for immunosuppressive drugs, and avoid the effects of aging.
Proponents of reproductive cloning believe that parents who cannot procreate should have access to technology.
Those who oppose cloning fear that the technology is not yet developed enough to be safe and that it could be prone to abuse (leading to the generation of humans from which organs and tissues would be removed), as well as the way of cloning them people can integrate with families and with society in general.
Religious groups are divided, and some oppose technology that usurps “God’s place” and, insofar as embryos are used, destroys a human life; others support the potentially life-saving benefits of therapeutic cloning.
Animal groups oppose the cloning of animals because of the number of cloned animals that suffer from malformations before they die.
Although the U.S. Food and Drug Administration has approved food from cloned animals, food-conscious groups oppose its use for safety.
Cloning of extinct and endangered species
The cloning, or more precisely, the reconstruction of the functional DNA of extinct species, has been, for decades, a dream. The possible implications were dramatized in the 1984 novel Carnosaurus and the 1990 novel Jurassic Park.
The best current cloning techniques have an average success rate of 9.4 percent (and up to 25 percent) when working with familiar species such as mice while cloning wild animals is typically less than 1 percent successful.
Several tissue banks, including the “frozen zoo” at the San Diego Zoo, have been created to store frozen tissue from the world’s rarest and endangered species.
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 from another species) could 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 the cheetah.
In 2002, geneticists at the Australian Museum announced that they had replicated the thylacine (Tasmanian tiger) DNA at the time it was extinct for about 65 years, using the polymerase chain reaction.
However, on February 15, 2005, the museum announced that it was stopping the project after tests showed that the specimens’ DNA (DNA) had too degraded by the preservative (ethanol).
On May 15, 2005, the thylacine project was announced to resume, with new participation from researchers in New South Wales and Victoria.
In 2003, for the first time, an extinct animal, the aforementioned Pyrenean ibex, was cloned at the Center for Food Technology and Research in Aragon, using the preserved nucleus of frozen cells from 2001 skin samples and egg cells from domestic goat.
The mountain goat died shortly after birth due to physical defects in its lungs.
One of the most anticipated targets for cloning was once the woolly mammoth but attempted to extract DNA from frozen mammoths have been unsuccessful. However, a joint Russian-Japanese team is currently working towards this goal.
In January 2011, Yomiuri Shimbun reported that a team of scientists led by Akira Iritani from Kyoto University had built on Dr. Wakayama’s research, saying they would extract DNA from a mammoth that had been preserved in a Russian laboratory and insert it into the ovules of an African elephant hoping to produce a mammoth embryo.
The researchers said they expected to produce a baby mammoth within six years. However, if possible, it was noted that the result would be an elephant-mammoth hybrid rather than an actual mammoth.
Another problem is the survival of the reconstructed mammoth: ruminants depend on a symbiosis with a specific microbiota in the stomach for digestion.
Scientists from the University of Newcastle and the University of New South Wales announced in March 2013 that the newly hatched gastric-hatching frog would be the subject of a cloning attempt to renew the species.
Many of these “extinction” projects are described in the Long Now Foundation’s Revive and Restore project.
Life expectancy
After an eight-year project involving a pioneering cloning technique, Japanese researchers created 25 generations of healthy cloned mice with an average lifespan, demonstrating that clones are not inherently shorter-lived than animals—born naturally.
Other sources have noted that the offspring of the clones tend to be healthier than the original clones and indistinguishable from naturally produced animals.
Dolly, the sheep was created from a sample of six-year-old cells from a mammary gland. Because of this, it aged faster than other naturally born animals because it started with cells that were already aging.
He died prematurely at the age of six, not only because of his age but also from respiratory problems and severe arthritis.
A detailed study published in 2016 and less clear studies by others suggests that once cloned animals are past the first month or two of life, they are generally healthy.
However, early pregnancy and neonatal losses are more fabulous with cloning than natural conception or assisted reproduction (in vitro fertilization). Current research is trying to overcome these problems.
In popular culture
The discussion of cloning in the popular media often presents the issue negatively.
In the Times newspaper of November 8, 1993, cloning was portrayed negatively, modifying Michelangelo’s Creation of Adam to represent Adam with five identical hands.
The March 10, 1997 issue of Newsweek also criticized the ethics of human cloning and included a graphic depicting identical babies in beakers.
The concept of cloning, particularly human cloning, has featured in many science fiction works.
A fictitious initial representation of cloning is the Bokanovsky Process, which appears in Aldous Huxley’s dystopian 1931 novel Brave New World.
The process is applied to human eggs fertilized in vitro, which causes them to divide into identical genetic copies of the original.
Following a renewed interest in cloning in the 1950s, the subject was explored in works such as Poul Anderson’s 1953 story, UN-Man, describing a technology called “exogenesis,” and Gordon Rattray Taylor’s book, The Biological Time Bomb, which popularized the term »cloning« in 1963.
Cloning is a recurring theme in several contemporary science fiction films, ranging from action movies such as Jurassic Park (1993), Alien Resurrection (1997), The 6th Day (2000), Resident Evil (2002), Star Wars: Episode II (2002) and The Island (2005), to comedies such as the 1973 Woody Allen film Sleeper.
The cloning process is variously represented in fiction. Many words describe the artificial creation of humans as a method of growing cells from a sample of tissue or DNA.
Replication can be instantaneous or take place through the slow growth of human embryos in artificial wombs.
In the long-running British television series Doctor Who, the Fourth Doctor and his partner Leela were cloned in seconds from DNA samples.
The clones in this story are short-lived and can only survive a few minutes before they expire.
Sci-fi movies like The Matrix and Star Wars: Episode II: Attack of the Clones have featured scenes of human fetuses being grown on an industrial scale in mechanical tanks.
The cloning of humans from body parts is also a common theme in science fiction.
Cloning stands out firmly among the sci-fi conventions parodied in Woody Allen’s Sleeper, whose plot centers on an attempt to clone a murdered dictator from his disembodied nose.
In the 2008 Doctor Who story “Journey’s End,” a duplicate version of the Tenth Doctor grows spontaneously from his hand, which had been cut off in a sword fight during a previous episode.
Cloning and identity
Science fiction has used cloning, more commonly and specifically human cloning, to raise controversial questions of identity.
A Number is a 2002 play by English playwright Caryl Churchill that addresses the issue of human cloning and identity, especially nature and nurture.
The story, set shortly, 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 former.
Caryl Churchill adapted one number for television in a co-production between the BBC and HBO Films.
In 2012, a Japanese television series called “Bunshin” was created. The story’s main character, Mariko, is a woman who studies child welfare in Hokkaido.
She always grew doubtful about the love of her mother, who was nothing like her and who died nine years earlier.
One day, she finds some of her mother’s belongings at a relative’s house and heads to Tokyo to search for the truth behind her birth. He later found out that it was a clone.
In the 2013 TV series Orphan Black, cloning is used as a scientific study on adapting the behavior of clones. Similarly, the book The Double by Nobel Prize winner José Saramago explores the emotional experience of a man who discovers that he is a clone.
Cloning as resurrection
Cloning has been used in fiction as a way to recreate historical figures. In Ira Levin’s 1976 novel The Boys from Brazil and its 1978 film adaptation, Josef Mengele uses cloning to create copies of Adolf Hitler.
In Michael Crichton’s 1990 novel Jurassic Park, which spawned a series of Jurassic Park feature films, a bioengineering company develops a technique to resurrect extinct species of dinosaurs by creating cloned creatures using DNA extracted from fossils.
Cloned dinosaurs are used to populate Jurassic Park for the entertainment of visitors. The scheme fails disastrously when the dinosaurs escape their enclosures.
Despite being selectively cloned as females to prevent them from reproducing, dinosaurs develop the ability to produce through parthenogenesis.
Cloning for War
The use of cloning for military purposes has also been explored in various fictional works. In Doctor Who, an alien race of armored warrior beings called the Sontarans was introduced in the 1973 series “The Time Warrior.”
Sontarans are depicted as squat, bald creatures that have been genetically engineered for combat. Its weak point is a “probiotic hole,” a small hole in the back of the neck associated with the cloning process.
The concept of cloned soldiers raised for combat was revised in “The Doctor’s Daughter” (2008) when the Doctor’s DNA is used to create a warrior named Jenny.
The 1977 movie Star Wars takes place in the context 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 armed clone troopers leading to the founding of the Galactic Empire.
Cloned soldiers are “manufactured” on an industrial scale, genetically conditioned for obedience and combat efficiency. 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.
Cloning has appeared in many video games. In Metal Gear Solid, the characters Solid Snake and Liquid Snake were born in a secret project as cloned soldiers.
In Halo, the cloning technology shows the recreation of organs. The Halo United Nations Faction Space Command uses cloning to train super soldiers when abducting children.
Here, non-clone children are trained as soldiers while clones covertly replace children abducted from the home.
Cloning for exploitation
A recurring subtopic of fictional cloning is the use of clones as a source of organs for transplantation.
Kazuo Ishiguro’s novel Never Let Me Go, and the film adaptation are set in an alternate history in which cloned humans are created to provide organ donations to naturally born humans.
The 2005 film The Island revolves around a similar plot, except that the clones are unaware of the reason for their existence.
In Raymond Han’s 2017 novel The Mind Clones Trilogy, a terminally ill dictator attempted to implant his mental clone into his son’s mind so that he could continue to rule the country.
Elsewhere in the trilogy, the usurpers conspired to replace the Chinese Politburo Standing Committee members using similar human clones.
The exploitation of human clones for dangerous and undesirable jobs was examined in the 2009 British science fiction film Luna.
In the futuristic novel Cloud Atlas and the subsequent film, one storyline centers on a genetically engineered clone named Sonmi ~ 451, one of the millions raised on an artificial “womb tank,” destined to serve from birth.
She is one of the thousands created for manual and emotional work; Sonmi herself works as a server in a restaurant. He later discovers that the only food source for clones, called ‘Soap,’ is made from the clones themselves.
