Cloning is the process of producing individual organisms with identical or virtually identical DNA, either by natural or artificial means. In nature, some organisms produce clones through asexual reproduction. In the field of biotechnology, cloning is the process of creating cloned organisms (copies) of cells and of DNA fragments (molecular cloning).
Etymology
Coined by Herbert J. Webber, the term clone derives from the Ancient Greek word κλών (klōn), twig, which is the process whereby a new plant is created from a twig. In botany, the term lusus was used.[1] In horticulture, the spelling clon was used until the early twentieth century; the final e came into use to indicate the vowel is a "long o" instead of a "short o".[2][3] Since the term entered the popular lexicon in a more general context, the spelling clone has been used exclusively.
Natural cloning
Cloning is a natural form of reproduction that has allowed life forms to spread for hundreds of millions of years. It is a reproduction method used by plants, fungi, and bacteria, and is also the way that clonal colonies reproduce themselves.[4][5] Examples of these organisms include blueberry plants, Hazel trees, the Pando trees,[6][7] the Kentucky coffeetree, Myrica, and the American sweetgum.
Molecular cloning
Molecular cloning refers to the process of making multiple molecules. Cloning is commonly used to amplify DNA fragments containing whole genes, but it can also be used to amplify any DNA sequence such as promoters, non-coding sequences and randomly fragmented DNA. It is used in a wide array of biological experiments and practical applications ranging from genetic fingerprinting to large scale protein production. Occasionally, the term cloning is misleadingly used to refer to the identification of the chromosomal location of a gene associated with a particular phenotype of interest, such as in positional cloning. In practice, localization of the gene to a chromosome or genomic region does not necessarily enable one to isolate or amplify the relevant genomic sequence. To amplify any DNA sequence in a living organism, that sequence must be linked to an origin of replication, which is a sequence of DNA capable of directing the propagation of itself and any linked sequence. However, a number of other features are needed, and a variety of specialised cloning vectors (small piece of DNA into which a foreign DNA fragment can be inserted) exist that allow protein production, affinity tagging, single-stranded RNA or DNA production and a host of other molecular biology tools.
Cloning of any DNA fragment essentially involves four steps[8]
- fragmentation - breaking apart a strand of DNA
- ligation – gluing together pieces of DNA in a desired sequence
- transfection – inserting the newly formed pieces of DNA into cells
- screening/selection – selecting out the cells that were successfully transfected with the new DNA
Although these steps are invariable among cloning procedures a number of alternative routes can be selected; these are summarized as a cloning strategy.
Initially, the DNA of interest needs to be isolated to provide a DNA segment of suitable size. Subsequently, a ligation procedure is used where the amplified fragment is inserted into a vector (piece of DNA). The vector (which is frequently circular) is linearised using restriction enzymes, and incubated with the fragment of interest under appropriate conditions with an enzyme called DNA ligase. Following ligation, the vector with the insert of interest is transfected into cells. A number of alternative techniques are available, such as chemical sensitisation of cells, electroporation, optical injection and biolistics. Finally, the transfected cells are cultured. As the aforementioned procedures are of particularly low efficiency, there is a need to identify the cells that have been successfully transfected with the vector construct containing the desired insertion sequence in the required orientation. Modern cloning vectors include selectable antibiotic resistance markers, which allow only cells in which the vector has been transfected, to grow. Additionally, the cloning vectors may contain colour selection markers, which provide blue/white screening (alpha-factor complementation) on X-gal medium. Nevertheless, these selection steps do not absolutely guarantee that the DNA insert is present in the cells obtained. Further investigation of the resulting colonies must be required to confirm that cloning was successful. This may be accomplished by means of PCR, restriction fragment analysis and/or DNA sequencing.
Cell cloning
Cloning unicellular organisms
Cloning a cell means to derive a population of cells from a single cell. In the case of unicellular organisms such as bacteria and yeast, this process is remarkably simple and essentially only requires the inoculation of the appropriate medium. However, in the case of cell cultures from multi-cellular organisms, cell cloning is an arduous task as these cells will not readily grow in standard media.
A useful tissue culture technique used to clone distinct lineages of cell lines involves the use of cloning rings (cylinders).[9] In this technique a single-cell suspension of cells that have been exposed to a mutagenic agent or drug used to drive selection is plated at high dilution to create isolated colonies, each arising from a single and potentially clonal distinct cell. At an early growth stage when colonies consist of only a few cells, sterile polystyrene rings (cloning rings), which have been dipped in grease, are placed over an individual colony and a small amount of trypsin is added. Cloned cells are collected from inside the ring and transferred to a new vessel for further growth.
Cloning stem cells
Somatic-cell nuclear transfer, popularly known as SCNT, can also be used to create embryos for research or therapeutic purposes. The most likely purpose for this is to produce embryos for use in stem cell research. This process is also called "research cloning" or "therapeutic cloning". The goal is not to create cloned human beings (called "reproductive cloning"), but rather to harvest stem cells that can be used to study human development and to potentially treat disease. While a clonal human blastocyst has been created, stem cell lines are yet to be isolated from a clonal source.[10]
Therapeutic cloning is achieved by creating embryonic stem cells in the hopes of treating diseases such as diabetes and Alzheimer's. The process begins by removing the nucleus (containing the DNA) from an egg cell and inserting a nucleus from the adult cell to be cloned.[11] In the case of someone with Alzheimer's disease, the nucleus from a skin cell of that patient is placed into an empty egg. The reprogrammed cell begins to develop into an embryo because the egg reacts with the transferred nucleus. The embryo will become genetically identical to the patient.[11] The embryo will then form a blastocyst which has the potential to form/become any cell in the body.[12]
The reason why SCNT is used for cloning is because somatic cells can be easily acquired and cultured in the lab. This process can either add or delete specific genomes of farm animals. A key point to remember is that cloning is achieved when the oocyte maintains its normal functions and instead of using sperm and egg genomes to replicate, the donor's somatic cell nucleus is inserted into the oocyte.[13] The oocyte will react to the somatic cell nucleus, the same way it would to a sperm cell's nucleus.[13]
The process of cloning a particular farm animal using SCNT is relatively the same for all animals. The first step is to collect the somatic cells from the animal that will be cloned. The somatic cells could be used immediately or stored in the laboratory for later use.[13] The hardest part of SCNT is removing maternal DNA from an oocyte at metaphase II. Once this has been done, the somatic nucleus can be inserted into an egg cytoplasm.[13] This creates a one-cell embryo. The grouped somatic cell and egg cytoplasm are then introduced to an electrical current.[13] This energy will hopefully allow the cloned embryo to begin development. The successfully developed embryos are then placed in surrogate recipients, such as a cow or sheep in the case of farm animals.[13]
SCNT is seen as a good method for producing agriculture animals for food consumption. It successfully cloned sheep, cattle, goats, and pigs. Another benefit is SCNT is seen as a solution to clone endangered species that are on the verge of going extinct.[13] However, stresses placed on both the egg cell and the introduced nucleus can be enormous, which led to a high loss in resulting cells in early research. For example, the cloned sheep Dolly was born after 277 eggs were used for SCNT, which created 29 viable embryos. Only three of these embryos survived until birth, and only one survived to adulthood.[14] As the procedure could not be automated, and had to be performed manually under a microscope, SCNT was very resource intensive. The biochemistry involved in reprogramming the differentiated somatic cell nucleus and activating the recipient egg was also far from being well understood. However, by 2014 researchers were reporting cloning success rates of seven to eight out of ten[15] and in 2016, a Korean Company Sooam Biotech was reported to be producing 500 cloned embryos per day.[16]
In SCNT, not all of the donor cell's genetic information is transferred, as the donor cell's mitochondria that contain their own mitochondrial DNA are left behind. The resulting hybrid cells retain those mitochondrial structures which originally belonged to the egg. As a consequence, clones such as Dolly that are born from SCNT are not perfect copies of the donor of the nucleus.
Organism cloning
Organism cloning (also called reproductive cloning) refers to the procedure of creating a new multicellular organism, genetically identical to another. In essence this form of cloning is an asexual method of reproduction, where fertilization or inter-gamete contact does not take place. Asexual reproduction is a naturally occurring phenomenon in many species, including most plants and some insects. Scientists have made some major achievements with cloning, including the asexual reproduction of sheep and cows. There is a lot of ethical debate over whether or not cloning should be used. However, cloning, or asexual propagation,[17] has been common practice in the horticultural world for hundreds of years.
Horticultural
The term clone is used in horticulture to refer to descendants of a single plant which were produced by vegetative reproduction or apomixis. Many horticultural plant cultivars are clones, having been derived from a single individual, multiplied by some process other than sexual reproduction.[18] As an example, some European cultivars of grapes represent clones that have been propagated for over two millennia. Other examples are potato and banana.[19]
Grafting can be regarded as cloning, since all the shoots and branches coming from the graft are genetically a clone of a single individual, but this particular kind of cloning has not come under ethical scrutiny and is generally treated as an entirely different kind of operation.
Many trees, shrubs, vines, ferns and other herbaceous perennials form clonal colonies naturally. Parts of an individual plant may become detached by fragmentation and grow on to become separate clonal individuals. A common example is in the vegetative reproduction of moss and liverwort gametophyte clones by means of gemmae. Some vascular plants e.g. dandelion and certain viviparous grasses also form seeds asexually, termed apomixis, resulting in clonal populations of genetically identical individuals.
Parthenogenesis
Clonal derivation exists in nature in some animal species and is referred to as parthenogenesis (reproduction of an organism by itself without a mate). This is an asexual form of reproduction that is only found in females of some insects, crustaceans, nematodes,[20] fish (for example the hammerhead shark[21]), and lizards including the Komodo dragon[21] 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
Artificial cloning of organisms may also be called reproductive cloning.
First steps
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.[22]
Methods
Reproductive cloning generally uses "somatic cell nuclear transfer" (SCNT) to create animals that are genetically identical. This process entails the transfer of a nucleus from a donor adult cell (somatic cell) to an egg from which the nucleus has been removed, or to a cell from a blastocyst from which the nucleus has been removed.[23] If the egg begins to divide normally it is transferred into the uterus of the surrogate mother. Such clones are not strictly identical since the somatic cells may contain mutations in their nuclear DNA. Additionally, the mitochondria in the cytoplasm also contains DNA and during SCNT this mitochondrial DNA is wholly from the cytoplasmic donor's egg, thus the mitochondrial genome is not the same as that of the nucleus donor cell from which it was produced. This may have important implications for cross-species nuclear transfer in which nuclear-mitochondrial incompatibilities may lead to death.
Artificial embryo splitting or embryo twinning, a technique that creates monozygotic twins from a single embryo, is not considered in the same fashion as other methods of cloning. During that procedure, a donor 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.[24] If both embryos are successful, it gives rise to monozygotic (identical) twins.
Dolly the sheep
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.[25] 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.[26] The embryo was then placed inside a female sheep that went through a normal pregnancy.[27] 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.[28] Her stuffed remains were placed at Edinburgh's Royal Museum, part of the National Museums of Scotland.[29]
Dolly was publicly significant because the effort showed that genetic material from a specific adult cell, designed to express only a distinct subset of its genes, can be redesigned to grow an entirely new organism. Before this demonstration, it had been shown by John Gurdon that nuclei from differentiated cells could give rise to an entire organism after transplantation into an enucleated egg.[30] However, this concept was not yet demonstrated in a mammalian system.
The first mammalian cloning (resulting in Dolly) had a success rate of 29 embryos per 277 fertilized eggs, which produced three lambs at birth, one of which lived. In a bovine experiment involving 70 cloned calves, one-third of the calves died quite young. The first successfully cloned horse, Prometea, took 814 attempts. Notably, although the first[clarification needed] clones were frogs, no adult cloned frog has yet been produced from a somatic adult nucleus donor cell.
There were early claims that Dolly had pathologies resembling accelerated aging. Scientists speculated that Dolly's death in 2003 was related to the shortening of telomeres, DNA-protein complexes that protect the end of linear chromosomes. However, other researchers, including Ian Wilmut who led the team that successfully cloned Dolly, argue that Dolly's early death due to respiratory infection was unrelated to problems with the cloning process. This idea that the nuclei have not irreversibly aged was shown in 2013 to be true for mice.[31]
Dolly was named after performer Dolly Parton because the cells cloned to make her were from a mammary gland cell, and Parton is known for her ample cleavage.[32]
Species cloned
The modern cloning techniques involving nuclear transfer have been successfully performed on several species. Notable experiments include:
- Tadpole: (1952) Robert Briggs and Thomas J. King had successfully cloned northern leopard frogs: thirty-five complete embryos and twenty-seven tadpoles from one-hundred and four successful nuclear transfers.[33][34]
- Carp: (1963) In China, embryologist Tong Dizhou produced the world's first cloned fish by inserting the DNA from a cell of a male carp into an egg from a female carp. He published the findings in a Chinese science journal.[35]
- Zebrafish: The first vertebrate cloned (1981) by George Streisinger (Streisinger, George; Walker, C.; Dower, N.; Knauber, D.; Singer, F. (1981), "Production of clones of homozygous diploid zebra fish (Brachydanio rerio)", Nature, 291 (5813): 293–296, Bibcode:1981Natur.291..293S, doi:10.1038/291293a0, PMID 7248006, S2CID 4323945)
- Sheep: Marked the first mammal being cloned (1984) from early embryonic cells by Steen Willadsen. Megan and Morag[36] cloned from differentiated embryonic cells in June 1995 and Dolly from a somatic cell in 1996.[37][35]
- Mice: (1986) A mouse was successfully cloned from an early embryonic cell. Soviet scientists Chaylakhyan, Veprencev, Sviridova, and Nikitin had the mouse "Masha" cloned. Research was published in the magazine Biofizika volume ХХХII, issue 5 of 1987.[clarification needed][38][39]
- Rhesus monkey: Tetra (January 2000) from embryo splitting and not nuclear transfer. More akin to artificial formation of twins.[40][41]
- Pig: the first cloned pigs (March 2000).[42] By 2014, BGI in China was producing 500 cloned pigs a year to test new medicines.[43]
- Gaur: (2001) was the first endangered species cloned.[44]
- Cattle: Alpha and Beta (males, 2001) and (2005) Brazil[45]
- Cat: CopyCat "CC" (female, late 2001), Little Nicky, 2004, was the first cat cloned for commercial reasons[46]
- Rat: Ralph, the first cloned rat (2003)[47]
- Mule: Idaho Gem, a john mule born 4 May 2003, was the first horse-family clone.[48]
- Horse: Prometea, a Haflinger female born 28 May 2003, was the first horse clone.[49]
- Dog: Snuppy, a male Afghan hound was the first cloned dog (2005).[50] In 2017, the world's first gene-editing clone dog, Apple, was created by Sinogene Biotechnology.[51]
- Wolf: Snuwolf and Snuwolffy, the first two cloned female wolves (2005).[52]
- Water buffalo: Samrupa was the first cloned water buffalo. It was born on 6 February 2009, at India's Karnal National Diary Research Institute but died five days later due to lung infection.[53]
- Pyrenean ibex (2009) was the first extinct animal to be cloned back to life; the clone lived for seven minutes before dying of lung defects.[54][55]
- Camel: (2009) Injaz, was the first cloned camel.[56]
- Pashmina goat: (2012) Noori, is the first cloned pashmina goat. Scientists at the faculty of veterinary sciences and animal husbandry of Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir successfully cloned the first Pashmina goat (Noori) using the advanced reproductive techniques under the leadership of Riaz Ahmad Shah.[57]
- Goat: (2001) Scientists of Northwest A&F University successfully cloned the first goat which use the adult female cell.[58]
- Gastric brooding frog: (2013) The gastric brooding frog, Rheobatrachus silus, thought to have been extinct since 1983 was cloned in Australia, although the embryos died after a few days.[59]
- Macaque monkey: (2017) First successful cloning of a primate species using nuclear transfer, with the birth of two live clones named Zhong Zhong and Hua Hua. Conducted in China in 2017, and reported in January 2018.[60][61][62][63] In January 2019, scientists in China reported the creation of five identical cloned gene-edited monkeys, using the same cloning technique that was used with Zhong Zhong and Hua Hua and Dolly the sheep, and the gene-editing Crispr-Cas9 technique allegedly used by He Jiankui in creating the first ever gene-modified human babies Lulu and Nana. The monkey clones were made to study several medical diseases.[64][65]
- Black-footed ferret: (2020) In 2020, a team of scientists cloned a female named Willa, who died in the mid-1980s and left no living descendants. Her clone, a female named Elizabeth Ann, was born on December 10. Scientists hope that the contribution of this individual will alleviate the effects of inbreeding and help black-footed ferrets better cope with plague. Experts estimate that this female's genome contains three times as much genetic diversity as any of the modern black-footed ferrets.[66]
Human cloning
Human cloning is the creation of a genetically identical copy of a human. The term is generally used to refer to artificial human cloning, which is the reproduction of human cells and tissues. It does not refer to the natural conception and delivery of identical twins. The possibility of human cloning has raised controversies. These ethical concerns have prompted several nations to pass legislation regarding human cloning and its legality. As of right now, scientists have no intention of trying to clone people and they believe their results should spark a wider discussion about the laws and regulations the world needs to regulate cloning.[67]
Two commonly discussed types of theoretical human cloning are therapeutic cloning and reproductive cloning. Therapeutic cloning would involve cloning cells from a human for use in medicine and transplants, and is an active area of research, but is not in medical practice anywhere in the world, as of 2021. 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.[68]
Ethical issues of cloning
There are a variety of ethical positions regarding the possibilities of cloning, especially human cloning. While many of these views are religious in origin, the questions raised by cloning are faced by secular perspectives as well. Perspectives on human cloning are theoretical, as human therapeutic and reproductive cloning are not commercially used; animals are currently cloned in laboratories and in livestock production.
Advocates support development of therapeutic cloning to generate tissues and whole organs to treat patients who otherwise cannot obtain transplants,[69] to avoid the need for immunosuppressive drugs,[68] and to stave off the effects of aging.[70] Advocates for reproductive cloning believe that parents who cannot otherwise procreate should have access to the technology.[71]
Opponents of cloning have concerns that technology is not yet developed enough to be safe[72] and that it could be prone to abuse (leading to the generation of humans from whom organs and tissues would be harvested),[73][74] as well as concerns about how cloned individuals could integrate with families and with society at large.[75][76]
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.[77][78]
Cloning of animals is opposed by animal-groups due to the number of cloned animals that suffer from malformations before they die, and while food from cloned animals has been approved by the US FDA,[79][80] its use is opposed by groups concerned about food safety.[81][82]
Cloning extinct and endangered species
Cloning, or more precisely, the reconstruction of functional DNA from extinct species has, for decades, been a dream. Possible implications of this were dramatized in the 1984 novel Carnosaur and the 1990 novel Jurassic Park.[83][84] The best current cloning techniques have an average success rate of 9.4 percent[85] (and as high as 25 percent[31]) when working with familiar species such as mice,[note 1] while cloning wild animals is usually less than 1 percent successful.[88]
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.[83][89][90] This is also referred to as "Conservation cloning".[91][92]
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.[93][94][95][96]
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.[97] 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.[98]
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.[99]
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.[100][101] It was noted, however that the result, if possible, would be an elephant-mammoth hybrid rather than a true mammoth.[102] Another problem is the survival of the reconstructed mammoth: ruminants rely on a symbiosis with specific microbiota in their stomachs for digestion.[102]
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.[103]
Many such "De-extinction" projects are described in the Long Now Foundation's Revive and Restore Project.[104]
Lifespan
After an eight-year project involving the use of a pioneering cloning technique, Japanese researchers created 25 generations of healthy cloned mice with normal lifespans, demonstrating that clones are not intrinsically shorter-lived than naturally born animals.[31][105] Other sources have noted that the offspring of clones tend to be healthier than the original clones and indistinguishable from animals produced naturally.[106]
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.[107][dubious ]
A detailed study released in 2016 and less detailed studies by others suggest that once cloned animals get past the first month or two of life they are generally healthy. However, early pregnancy loss and neonatal losses are still greater with cloning than natural conception or assisted reproduction (IVF). Current research is attempting to overcome these problems.[32]