From Wikipedia, the free encyclopedia
The term clone
is derived from κλών
, the Greek
word for "trunk, branch", 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.
Molecular cloning refers to the process of making multiple copies of a defined DNA
sequence. Cloning is frequently 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
. 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 (α-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 cell-line colonies using cloning rings
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)
. 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 clonally 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.
(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 mean all descendants of a single plant, produced by vegetative reproduction
. 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
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.
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
Dolly the Sheep
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 entire 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 237 eggs were used to create 29 embryos, which only produced three lambs at birth, only one of which lived. Seventy calves have been created from 9,000 attempts and one third of them died young; Prometea
took 328 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.
Though Dolly was the first cloned mammal, the first vertebrate to be cloned was a tadpole
in 1952 
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 successfully cloned mammal. 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.
- 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.
- Camel: (2009) Injaz, is the first cloned camel.
- Pyrenean Ibex : (2009) A cloned individual of allows this subspecies to become un-extinct for 7 minutes before the individual dies, and the subspecies becomes re-extinct.
Main article: Human cloning
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 human hybrid human clone was created in November 1998, by American 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
Although the practice of cloning organisms has been widespread for several thousands of years in the form of horticultural cloning, the recent technological advancements that have allowed for cloning of animals (and potentially humans) have been highly controversial. The Catholic Church and many religious organizations oppose all forms of cloning, on the grounds that life begins at conception. Conversely, Judaism does not equate life with conception and, though some question the wisdom of cloning, Orthodox
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.
is a scientist and outspoken critic against restrictions on cloning research.
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.
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.
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
) were frozen immediately after it died. Researchers are also considering cloning endangered species such as the giant panda, ocelot, 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), extinct about 65 years previous, 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. Most recently, on May 15, 2005 it was announced that the Thylacine project would be revived, with new participation from researchers in New South Wales
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. Essentially, if cloning an extinct species were successful — it must be considered that cloning is still an experimental technology that succeeds only by chance — it is more likely than not that any resulting animals, even if they were healthy, would be little more than curios or museum pieces.
Cloning endangered species is a highly ideological issue. Many conservation biologists
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
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:
Vertebrate cloning poses little risk to the environment, but it can consume scarce conservation resources, and its chances of success in preserving species seem poor. To date, the conservation benefits of transgenics and vertebrate cloning remain entirely theoretical, but many of the risks are known and documented. Conservation biologists should devote their research and energies to the established methods of conservation, none of which require transgenics or vertebrate cloning.
- ^ Peter J. Russel (2005). iGenetics: A Molecular Approach. San Francisco, California, United States of America: Pearson Education. ISBN 0-8053-4665-1.
- ^ McFarland, Douglas (2000). "Preparation of pure cell cultures by cloning". Methods in Cell Science 22 (1): 63–66. doi:10.1023/A:1009838416621. PMID 10650336.
- ^ Gil, Gideon (2008-01-17). "California biotech says it cloned a human embryo, but no stem cells produced". Boston Globe. http://www.boston.com/news/health/blog/2008/01/california_biot.html.
- ^ Illmensee K, Levanduski M, Vidali A, Husami N, Goudas VT (February 2009). "Human embryo twinning with applications in reproductive medicine". Fertil. Steril.. doi:10.1016/j.fertnstert.2008.12.098. PMID 19217091.
- ^ BLOODLINES. Timeline
- ^ "Кто изобрел клонирование?". Archived from the original on 2004-12-23. http://web.archive.org/web/20041223221951/http://www.whoiswho.ru/russian/Curnom/22003/cl.htm. (Russian)
- ^ McLaren A (2000). "Cloning: pathways to a pluripotent future". Science 288 (5472): 1775–80. doi:10.1126/science.288.5472.1775. PMID 10877698.
- ^ CNN. Researchers clone monkey by splitting embryo 2000-01-13. Retrieved 2008-08-05
- ^ http://archives.cnn.com/2001/NATURE/01/12/cloned.gaur/index.html
- ^ Camacho, Keite. Embrapa clona raça de boi ameaçada de extinção. Agência Brasil. 2005-05-20. (Portuguese) Retrieved 2008-08-05
- ^ 
- ^ http://timesofindia.indiatimes.com/India-clones-worlds-first-buffalo/articleshow/4120044.cms
- ^ Spencer, Richard (April 14, 2009). "World's first cloned camel unveiled in Dubai". Telegraph.co.uk. http://www.telegraph.co.uk/news/newstopics/howaboutthat/5153780/Worlds-first-cloned-camel-unveiled-in-Dubai.html. Retrieved April 15, 2009.
- ^ Pence, Gregory E. (1998). Who’s Afraid of Human Cloning?. Rowman & Littlefield. paperback ISBN 0-8476-8782-1 and hardcover ISBN 0-8476-8781-3.
- ^ "AAAS Statement on Human Cloning". http://www.aaas.org/spp/sfrl/docs/cloningstatement.shtml.
- ^ citation needed
- ^ BBC News | SCI/TECH | Details of hybrid clone revealed
- ^ Mature Human Embryos Created From Adult Skin Cells Washingtonpost.com
- ^ Michael Brody. Avraham Steinberg.
- ^ New Page 0
- ^ 
- ^ Meat of Cloned Food is Safe to Eat, FDA Says
- ^ http://www.hsus.org/farm/resources/research/practices/genetic_engineering_and_cloning_farm_animals.html
- ^ http://www.centerforfoodsafety.org/pubs/FINAL_FORMATTEDprime%20time.pdf (PDF)
- ^ http://www.consumersunion.org/pdf/FDA_clone_comments.pdf (PDF)
- ^ http://ga3.org/campaign/CloneTracking
- ^ "Scientists 'to clone mammoth'". BBC News. 2003-08-18. http://news.bbc.co.uk/2/hi/asia-pacific/3075381.stm.
- ^ Heidi B. Perlman (2000-10-08). "Scientists Close on Extinct Cloning". Associated Press. http://www.washingtonpost.com/wp-srv/aponline/20001008/aponline171938_000.htm.
- ^ Pence, Gregory E. (2005). Cloning After Dolly: Who's Still Afraid?. Rowman & Littlefield. ISBN 0-7425-3408-1.
- ^ Holloway, Grant (2002-05-28). "Cloning to revive extinct species". CNN.com. http://www.cnn.com/2002/WORLD/asiapcf/auspac/05/28/aust.thylacines/.
- ^ a b Ehrenfeld, David (2006). "Transgenics and Vertebrate Cloning as Tools for Species Conservation". Conservation Biology 20 (3): 723–732. doi:10.1111/j.1523-1739.2006.00399.x. PMID 16909565.
External links and references