300px Cloning in biology is the process of producing similar populations of genetically identical individuals that occurs in nature when organisms such as bacteria, insects or plants reproduce asexually. Cloning in biotechnology refers to processes used to create copies of DNA fragments (molecular cloning), cells (cell cloning), or organisms. The term also refers to the production of multiple copies of a product such as digital media or software.
The term clone is derived from the Ancient Greek word (kl n, twig ), referring to the process whereby a new plant can be created from a twig. In horticulture, the spelling clon was used until the twentieth century; the final e came into use to indicate the vowel is a "long o" instead of a "short o". Since the term entered the popular lexicon in a more general context, the spelling clone has been used exclusively.
In the United States, the human consumption of meat and other products from cloned animals was approved by the FDA on December 28, 2006, with no special labeling required. Cloned beef and other products have since been regularly consumed in the US without distinction. Such practice has met strong resistance in other regions, such as Europe, particularly over the labeling issue.
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 expression, tagging, single stranded RNA and DNA production and a host of other manipulations.
Cloning of any DNA fragment essentially involves four steps
- 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 sensitivation 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.
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.
Cloning cell-line colonies using cloning rings
A useful tissue culture technique used to clone distinct lineages of cell lines involves the use of cloning rings (cylinders). According to 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 of 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 in stem cell research
Somatic cell nuclear transfer, 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.
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 taking out the nucleus that contains the DNA from an egg and putting it in a nucleus from an adult. In the case of someone with Alzheimer s disease, the nucleus from a skin cell of that patient is placed into an empty egg. The reprogrammed cell begins to develop into an embryo because the egg reacts with the transferred nucleus. The embryo will become genetically identical to the patient. The embryo will then form a blastocyst which has the potential to form/become any cell in the body.
The reason why SCNT is used for cloning is because somatic cells can be easily acquired and cultured in the lab. This process can either add or delete specific genomes of farm animals. A key point to remember is that cloning is achieved when the oocyte maintains its normal functions and instead of using sperm and egg genomes to replicate, the oocyte is inserted into the donor s somatic cell nucleus. The oocyte will react on the somatic cell nucleus, the same way it would on sperm cells.
SCNT Process The process of cloning a particular farm animal using SCNT is relatively the same for all animals. The first step is to collect the somatic cells from the animal that will be cloned. The somatic cells could be used immediately or stored in the laboratory for later use. The hardest part of SCNT is removing maternal DNA from an oocyte at metaphase II. Once this has been done, the somatic nucleus can be inserted into an egg cytoplasm. This creates a one-cell embryo. The grouped somatic cell and egg cytoplasm are then introduced to an electrical current. This energy will hopefully allow the cloned embryo to begin development. The successfully developed embryos are then placed in surrogate recipients, such as a cow or sheep in the case of farm animals.
Applications of SCNT SCNT is seen to be a great 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.
Limitations of SCNT Stresses placed on both the egg cell and the introduced nucleus are enormous, leading to a high loss in resulting cells. For example, Dolly the sheep was born after 277 eggs were used for SCNT, which created 29 viable embryos. Only three of these embryos survived until birth, and only one survived to adulthood. As the procedure currently cannot be automated, but has to be performed manually under a microscope, SCNT is very resource intensive. The biochemistry involved in reprogramming the differentiated somatic cell nucleus and activating the recipient egg is also far from understood.
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 (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 (see vegetative reproduction) 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, has been common practice in the horticultural world for hundreds of years.
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. As an example, some European cultivars of grapes represent clones that have been propagated for over two millennia. Other examples are potato and banana. 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. Parts of a large clonal colony often become detached from the parent, termed fragmentation, to form separate individuals. Some plants e.g. dandelion also form seeds asexually, termed apomixis, resulting in clonal populations of genetically identical individuals.
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 and lizards. 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.
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 that has no nucleus. 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 DNA is wholly from the donor 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 may also be used as a method of cloning, where an embryo is split in the maturation before embryo transfer. It is optimally performed at the 6- to 8-cell stage, where it can be used as an expansion of IVF to increase the number of available embryos. If both embryos are successful, it gives rise to monozygotic (identical) twins.
A blastocyst is formed in the early stage of the development of an embryo. During cloning process, the blastocyst cells often are obtained by scientists 5 days after the egg has been divided.
Dolly the Sheep
Dolly, a Finn-Dorset ewe, was the first mammal to have been successfully cloned from an adult cell. Dolly was formed by taking a cell from the udder of her biological mother. Her embryo was created by taking the cell and inserting it into a sheep ovum. The embryo was then placed inside a female sheep that went through a normal pregnancy. She was cloned at the Roslin Institute in Scotland and lived there from her birth in 1996 until her death in 2003 when she was six. Her stuffed remains were placed at Edinburgh's Royal Museum, part of the National Museums of Scotland.
Dolly was publicly significant because the effort showed that the genetic material from 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, it had been shown by John Gurdon that nuclei from differentiated cells could give rise to an entire organism after transplantation into an enucleated egg. However, this concept was not yet demonstrated in a mamallian system.
Cloning Dolly the sheep had a low success rate per fertilized egg; she was born after 277 eggs were used to create 29 embryos, which only produced three lambs at birth, only one of which lived. Seventy calves have been created and one third of them died young; Prometea took 277 attempts. Notably, although the first clones were frogs, no adult cloned frog has yet been produced from a somatic adult nucleus donor cell.
There were early claims that Dolly the Sheep 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 deficiencies with the cloning process.
On September 15, 2007, the Philippines announced its development of Southeast Asia s first cloned water buffalo. The Philippine Council for Agriculture, Forestry and Natural Resources Research and Development (PCARRD), under the Department of Science and Technology in Los Ba os, Laguna approved this project.
The modern cloning techniques involving nuclear transfer have been successfully performed on several species. Landmark experiments in chronological order:
- Tadpole: (1952) Many scientists questioned whether cloning had actually occurred and unpublished experiments by other labs were not able to reproduce the reported results.
- 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.
- Mice: (1986) A mouse was the first mammal 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 II, issue 5 of 1987.
- Sheep: (1996) From early embryonic cells by Steen Willadsen. Megan and Morag cloned from differentiated embryonic cells in June 1995 and Dolly the sheep from a somatic cell in 1997.
- Rhesus Monkey: Tetra (January 2000) from embryo splitting
Gaur: (2001) was the first endangered species cloned.
- Cattle: Alpha and Beta (males, 2001) and (2005) Brazil
Cat: CopyCat "CC" (female, late 2001), Little Nicky, 2004, was the first cat cloned for commercial reasons
- Dog: Snuppy, a male Afghan hound was the first cloned dog (2005).
- Rat: Ralph, the first cloned rat (2003)
- Mule: Idaho Gem, a john mule born 4 May 2003, was the first horse-family clone.
- Horse: Prometea, a Haflinger female born 28 May 2003, was the first horse clone.
- Water Buffalo: Samrupa was the first cloned water buffalo. It was born on February 6, 2009, at India's Karnal National Diary Research Institute but died five days later due to lung infection.
- Pyrenean Ibex (2009) was the first "extinct" animal (while the species is not extinct, nor even endangered, no living examples of the Pyrenean subspecies had been known since 2000) to be cloned back to life; the clone lived for seven minutes before dying of lung defects.
- Camel: (2009) Injaz, is the first cloned camel.
- Pashmina goat: (2012) Noorie, is the first cloned pashmina goat. Scientists at the faculty of veterinary sciences and animal husbandry of Sher-e-Kashmir University of Agriculture Sciences and Technology (SKUAST-Kashmir) successfully cloned the first Pashmina goat (Noorie) using the advanced reproductive techniques under the leadership of Riaz Ahmad Shah.
Human cloning is the creation of a genetically identical copy of an existing or previously existing human. The term is generally used to refer to artificial human cloning; human clones in the form of identical twins are commonplace, with their cloning occurring during the natural process of reproduction. There are two commonly discussed types of human cloning: therapeutic cloning and reproductive cloning. Therapeutic cloning involves cloning adult cells for use in medicine and is an active area of research. Reproductive cloning would involve making cloned humans. A third type of cloning called replacement cloning is a theoretical possibility, and would be a combination of therapeutic and reproductive cloning. Replacement cloning would entail the replacement of an extensively damaged, failed, or failing body through cloning followed by whole or partial brain transplant.
The various forms of human cloning are controversial. There have been numerous demands for all progress in the human cloning field to be halted. Most scientific, governmental and religious organizations oppose reproductive cloning. The American Association for the Advancement of Science (AAAS) and other scientific organizations have made public statements suggesting that human reproductive cloning be banned until safety issues are resolved. Serious ethical concerns have been raised by the future possibility of harvesting organs from clones. Some people have considered the idea of growing organs separately from a human organism - in doing this, a new organ supply could be established without the moral implications of harvesting them from humans. Research is also being done on the idea of growing organs that are biologically acceptable to the human body inside of other organisms, such as pigs or cows, then transplanting them to humans, a form of xenotransplantation.
The first hybrid human clone was created in November 1998, by Advanced Cell Technologies. It was created from a man's leg cell, and a cow's egg whose DNA was removed. It was destroyed after 12 days. Since a normal embryo implants at 14 days, Dr Robert Lanza, ACT's director of tissue engineering, told the Daily Mail newspaper that the embryo could not be seen as a person before 14 days. While making an embryo, which may have resulted in a complete human had it been allowed to come to term, according to ACT: "[ACT's] aim was 'therapeutic cloning' not 'reproductive cloning'"
On January, 2008, Wood and Andrew French, Stemagen's chief scientific officer in California, announced that they successfully created the first 5 mature human embryos using DNA from adult skin cells, aiming to provide a source of viable embryonic stem cells. Dr. Samuel Wood and a colleague donated skin cells, and DNA from those cells was transferred to human eggs. It is not clear if the embryos produced would have been capable of further development, but Dr. Wood stated that if that were possible, using the technology for reproductive cloning would be both unethical and illegal. The 5 cloned embryos, created in Stemagen Corporation lab, in La Jolla, were destroyed.
Ethical issues of cloning
Because of recent technological advancements, the cloning of animals (and potentially humans) has been an issue. The Catholic Church and many religious organizations oppose all forms of cloning, on the grounds that life begins at conception. Judaism does not equate life with conception and, though some question the wisdom of cloning, Orthodox Judaism rabbis generally find no firm reason in Jewish law and ethics to object to cloning. From the standpoint of classical liberalism, concerns also exist regarding the protection of the identity of the individual and the right to protect one's genetic identity.
Gregory Stock is a scientist and outspoken critic against restrictions on cloning research. Bioethicist Gregory Pence also attacks the idea of criminalizing attempts to clone humans.
The social implications of an artificial human production scheme were famously explored in Aldous Huxley's novel Brave New World.
On December 28, 2006, the U.S. Food and Drug Administration (FDA) approved the consumption of meat and other products from cloned animals. Cloned-animal products were said to be virtually indistinguishable from the non-cloned animals. Furthermore, companies would not be required to provide labels informing the consumer that the meat comes from a cloned animal.
Critics have raised objections to the FDA's approval of cloned-animal products for human consumption, arguing that the FDA's research was inadequate, inappropriately limited, and of questionable scientific validity. Several consumer-advocate groups are working to encourage a tracking program that would allow consumers to become more aware of cloned-animal products within their food.
Joseph Mendelson, legal director of the Center for Food Safety, said that cloned food still should be labeled since safety and ethical issues about it remain questionable.
Carol Tucker Foreman, director of food policy at the Consumer Federation of America, stated that FDA does not consider the fact that the results of some studies revealed that cloned animals have increased rates of mortality and deformity at birth.
Another concern is that the biotechnologies used on animals may someday be used on humans. Some people may be more open to the idea of cloning of animals because most western countries have passed legislation against cloning humans, yet only a few countries passed legislation against cloning animals.
Possible Abnormalities due to Cloning
Researchers have found several abnormalities in cloned organisms, particularly in mice. The cloned organism may be born normal and resemble its non-cloned counterpart, but majority of the time will express changes in its genome later on in life. The concern with cloning humans is that the changes in genomes may not only result in changes in appearance, but in psychological and personality changes as well. The theory behind this is that the biological blueprint of the genes is the same in cloned animals as it is in normal ones, but they are read and expressed incorrectly. DNA arrays were used to prove this claim in the research lab of Professor Rudolf Jaenisch. Jaenisch studied placentas from cloned mice and found that one in every 25 genes was expressed abnormally. Results of these abnormally expressed genes in the cloned mice were premature death, pneumonia, liver failure and obesity.
Cloning extinct and endangered species
Cloning, or more precisely, the reconstruction of functional DNA from extinct species has, for decades, been a dream of some scientists. The possible implications of this were dramatized in the best-selling novel by Michael Crichton and high budget Hollywood thriller Jurassic Park. In real life, 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. And 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 African elephant in hopes of producing a mammoth embryo. The researchers said they hoped to produce a baby mammoth within six years.
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. The "Frozen Zoo" at the San Diego Zoo now stores frozen tissue from the world's rarest and most endangered species.
In 2002, geneticists at the Australian Museum announced that they had replicated DNA of the Thylacine (Tasmanian Tiger), at the time extinct for about 65 years, using polymerase chain reaction. However, on February 15, 2005 the museum announced that it was stopping the project after tests showed the specimens' DNA had been too badly degraded by the (ethanol) preservative. On 15 May 2005 it was announced that the Thylacine project would be revived, with new participation from researchers in New South Wales and Victoria.
In January 2009, 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 DNA of the skin samples from 2001 and domestic goat egg-cells. (The ibex died shortly after birth due to physical defects in its lungs.)  One of the continuing obstacles in the attempt to clone extinct species is the need for nearly perfect DNA. Cloning from a single specimen could not create a viable breeding population in sexually reproducing animals. Furthermore, even if males and females were to be cloned, the question would remain open whether they would be viable at all in the absence of parents that could teach or show them their natural behavior.
Cloning endangered species is a highly ideological issue. Many conservation biologists and environmentalists vehemently oppose cloning endangered species mainly because they think it may deter donations to help preserve natural habitat and wild animal populations. The "rule-of-thumb" in animal conservation is that, if it is still feasible to conserve habitat and viable wild populations, breeding in captivity should not be undertaken in isolation.
In a 2006 review, David Ehrenfeld concluded that cloning in animal conservation is an experimental technology that, at its state in 2006, could not be expected to work except by pure chance and utterly failed a cost-benefit analysis. Furthermore, he said, it is likely to siphon funds from established and working projects and does not address any of the issues underlying animal extinction (such as habitat destruction, hunting or other overexploitation, and an impoverished gene pool). While cloning technologies are well-established and used on a regular basis in plant conservation, care must be taken to ensure genetic diversity. He concluded:
On the 7th of December 2011 it was announced that a team from the Siberian mammoth museum and Japan's Kinki University plan to clone a woolly mammoth from a well preserved sample of bone marrow found in August 2011. The team claim that the cloning could be complete within the next five years. However, others have expressed doubt about the feasibility of the experiment.
In science fiction
Cloning has been used in countless science fiction works throughout the years. Human cloning is usually most popular, mainly due to fact that it brings up controversial questions of identity. For example, in Aldous Huxley s prescient novel Brave New World (1932), human cloning is a major plot device that not only drives the story along but also makes the reader think critically about what identity means; this was picked up fifty years later in C. J. Cherryh s novels Forty Thousand in Gehenna (1983) and Cyteen (1988). Kazuo Ishiguro's 2005 novel Never Let Me Go centres on human clones and considers the ethics of the practice. Another book that embodies the ideas of cloning is "The House of the Scorpion" which explores the rights of human clones and organ harvesting, set from the eyes of a clone.
Star Wars portrays human cloning in Star Wars Episode II: Attack of the Clones and Star Wars Episode III: Revenge of the Sith, in the form of the Grand Army of the Republic, an army of clone troopers. The Expanded Universe also has numerous examples of cloning, including the Thrawn trilogy, The Hand of Thrawn duology, and Clone Wars-era media.
- The President's Council on Bioethics
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