Cloning

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Cloning is the process of creating an identical copy of an original organism or thing. Cloning in the biological sense results in a molecule, single cell (for example: bacteria, lymphocytes, etc.) or multi-cellular organism that has been directly copied from and is therefore genetically identical to the molecule, cells or organism being cloned. Sometimes this term can refer to "natural" clones, which may arise when an organism reproduces asexually by chance (as with identical twins), but in common parlance, a clone is an identical copy created intentionally.

The term clone is derived from κλων, the Greek word for "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.

Contents 1 Molecular cloning 2 Genetic cloning 3 Organism 3.1 Horticultural 3.2 Animals 3.3 Reproductive Cloning 3.3.1 Species cloned 3.3.2 Health aspects 3.3.3 Dolly the Sheep 3.3.4 Human cloning 3.3.5 Ethical issues of human cloning 3.3.6 Cloning extinct and endangered species 3.4 Embryo 4 References 5 External links and references

Molecular cloning

Main article: clone (genetics)

Molecular cloning refers to the procedure of isolating a DNA sequence of interest and obtaining multiple copies of it in an organism. Cloning is frequently employed to amplify DNA fragments containing genes, an essential step in their subsequent analysis. Frequently, 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, localisation of the gene to a chromosome or genomic region does not necessarily enable one to isolate or amplify the relevant genomic sequence.

Cloning of any DNA sequence involves the following four steps: fragmentation, ligation, transfection, and screening/selection. Initially, the DNA of interest needs to be fragmented to provide a relevant DNA segment of suitable size. Preparation of DNA fragments for cloning is frequently achieved by means of PCR, but it may also be accomplished by restriction enzyme digestion and sometimes fractionation by gel electrophoresis. Subsequently, a ligation procedure is employed whereby the amplified fragment is inserted into a vector. The vector (which is frequently circular) is linearised by means of 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. Most commonly electroporation is employed, although a number of alternative techniques are available, such as chemical sensitivation of cells. Finally, the transfected cells are cultured. As the aforementioned procedures are of particularly low efficiency, there is need to identify the cell colonies that have been successfully transfected with the vector construct containing the desired insertion sequence. 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 color selection markers which provide blue/white screening (α-factor complimentation) 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 is required to confirm that cloning was successful. This may be accomplished by means of PCR, restriction fragment analysis and DNA sequencing.

Genetic cloning

Cloning a cell means to derive a (clonal) population of cells from a single cell. This is an important in vitro procedure when the expansion of a single cell with certain characteristics is desired, for example in the production of gene-targeted ES cells. A valuable 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 which 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.

Most individuals began as a single cell and are therefore the result of clonal expansion in vivo.

Organism

Main article: Asexual reproduction

Cloning means to create a new organism with the same genetic information as a cell from an existing one(identical). It is an asexual method of reproduction, where fertilization or inter-gamete contact does not take place. Asexual reproduction (also known as agamogenesis) is a form of reproduction which does not involve meiosis, gamete formation, or fertilization. In laymen's terms, there is only one "parent" involved. This form of reproduction is common among simple organisms such as amoeba and other single-celled organisms, although most plants reproduce asexually as well (see vegetative reproduction).

Horticultural

The term clone is used in horticulture to mean all descendants of a single plant, 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, although the root systems may be genetically genuine examples of cloning in the broader biological sense, as they create genetically identical organisms by biological means, 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 also form seeds asexually, termed apomixis, e.g. dandelion.

Animals

Cloning exists in nature in some animal species and is referred to as parthenogenesis. An example is the "Little Fire Ant" (Wasmannia auropunctata), which is native to Central and South America but has spread throughout many tropical environments.

Reproductive Cloning

Reproductive cloning is a technology used to generate an animal that has the same nuclear DNA as another currently or previously existing animal. Dolly the sheep, was created by reproductive cloning technology. In a process called "somatic cell nuclear transfer" (SCNT), scientists transfer genetic material from the nucleus of a donor adult cell to an egg whose nucleus, and thus its genetic material, has been removed. The reconstructed egg containing the DNA from a donor cell must be treated with chemicals or electric current in order to stimulate cell division. Once the cloned embryo reaches a suitable stage, it is transferred to the uterus of a female host where it continues to develop until birth.

Dolly or any other animal created using nuclear transfer technology is not truly an identical clone of the donor animal. Only the clone's chromosomal or nuclear DNA is the same as the donor. Some of the clone's genetic materials come from the mitochondria in the cytoplasm of the enucleated egg. Mitochondria, which are organelles that serve as power sources to the cell, contain their own short segments of DNA, although this is only 0.01% of the total DNA. Acquired mutations in mitochondrial DNA are believed to play an important role in the aging process.

Also mutations occur with every cell division so no two cells in an individual are identical, nor are clones. Thus, nuclear transfer clones from different maternal lineages are not clones in the strictest sense because 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 inviability.

Species cloned

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 cloned a fish. He published the findings in an obscure Chinese science journal which was never translated into English.^[1]
• Mice: (1986) was the first successfully cloned mammal; Soviet scientists Chaylakhyan, Veprencev, Sviridova, Nikitin had mice "Masha" cloned. Research was published in the magazine "Biofizika" volume ХХХII, issue 5 of 1987.^[2]
• Sheep: (1996) From early embryonic cells by Steen Willadsen. Megan and Morag cloned from differentiated embryonic cells in June 1995 and Dolly the sheep in 1997.
• Rhesus Monkey: Tetra (female, January 2000) from embryo splitting
• Cattle: Alpha and Beta (males, 2001) and (2005) Brazil^[3]
• Cat: CopyCat "CC" (female, late 2001), Little Nicky, 2004, was the first cat cloned for commercial reasons
• Mule: Idaho Gem, a john mule born 2003-05-04, was the first horse-family clone.
• Horse: Prometea, a Haflinger female born 2003-05-28, was the first horse clone.

For a complete list see: List of animals that have been cloned. During the first several divisions of a fertilized egg, no differentiation occurs and the cells can be separated without harm, but each will grow into an identical individual. This process has been used on cattle for decades to produce hundreds of identical individuals in some cases. This process is not considered cloning, but is called budding. The new individual is not derived from a differentiated cell, but from an undifferentiated egg. There is no way to determine which are the clones and which is the original.

Health aspects

The success rate of cloning has been low: Dolly the sheep was born after 277 eggs were used to create 29 embryos, which only produced three lambs at birth, only one of which lived, Dolly. Seventy calves have been created from 9,000 attempts and one third of them died young; Prometea took 328 attempts, and, more recently, Paris Texas was created after 400 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 accelerated aging. Aging of this type is thought to be due to shortening of telomeres, regions at the tips of chromosomes which prevent genetic threads fraying every time a cell divides. Over time telomeres get worn down until cell-division is no longer possible — this is thought to be a cause of aging. However, subsequent studies showed that, if anything, Dolly's telomere were longer than normal.^{[verification needed]} Dolly died in the year of 2003. Ian Wilmut said that Dolly's early death had nothing to do with cloning but with a respiratory infection common to lambs raised indoors like Dolly.

Consistent with Dolly's telomeres being longer, analysis of the telomeres from cloned cows showed that they were also longer. This suggests clones could live longer life spans although many died young after excessive growth. Researchers think that this could eventually be developed to reverse aging in humans, provided that this is based chiefly on the shortening of telomeres. Although some work has been performed on telomeres and aging in nuclear transfer clones, the evidence is at an early stage. ^[4]

Dolly the Sheep

Main article: Dolly the Sheep

Dolly (1996-07-05 – 2003-02-14), a ewe, was the first mammal to have been successfully cloned from an adult cell (while the mice in USSR were cloned from embryo cell back in 1986[1]). She was cloned at the Roslin Institute in Scotland and lived there until her death when she was 6. Her birth was announced on 1997-02-22.

The name "Dolly" came from a suggestion by Jesse Haase who helped with her birth, in honor of Dolly Parton, because it was a mammary cell that was cloned. The technique that was made famous by her birth is somatic cell nuclear transfer, in which a non-reproductive cell containing a nucleus is placed in a de-nucleated ovum (which then develops into a fetus). When Dolly was cloned in 1996 from a cell taken from a six-year-old ewe, she became the center of much controversy that still exists today.

Dolly's success is truly remarkable because it proved that the genetic material from a specialized adult cell, such as an udder cell programmed to express only those genes needed by udder cells, could be reprogrammed to generate an entire new organism. Before this demonstration, scientists believed that once a cell became specialized as a liver, heart, udder, bone, or any other type of cell, the change was permanent and other unneeded genes in the cell would become inactive. Some scientists believe that errors or incompleteness in the reprogramming process cause the high rates of death, deformity, and disability observed among animal clones.

On 2003-04-09 her stuffed remains were placed at Edinburgh's Royal Museum, part of the National Museums of Scotland.

Ian Wilmut's role in cloning Dolly the sheep is in doubt. In 2006 he admitted under oath in a Scottish court that he did not clone Dolly the Sheep and was not responsible for the scientific breakthrough which made it all possible. [2] He credited Keith Campbell as being the brains behind Dolly the Sheep.

In August 2006, Iranian scientists oversaw the birth of the Middle East’s first cloned animal – a lamb that died minutes after it was born. However, in September 2006, Iranian scientists successfully cloned a sheep, by somatic cell nuclear transfer, at the Royan research institute in Isfahan, Iran’s second cloned lamb is still alive. [3]

Human cloning

Main article: Human cloning

Human cloning is the creation of a genetically identical copy of an existing, or previously existing human, by growing cloned tissue from that individual. 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.

Human cloning is amongst the most controversial forms of the practice. There have been numerous demands for all progress in the human cloning field to be halted. One of the most ethically questionable problems with human cloning is farming of organs from clones. For example, many believe it is unethical to use a human clone to save the life of another. In this scenario, the cloned human would be euthanized so that the vital organs could be harvested. This process of renewing the body's organs would potentially increase the life expectancy of a human by 50 years.

The cloning described above is reproductive cloning, not to be confused with research cloning in which only parts (such as an organ) are cloned using genetic material from a patient's tissues.

Ethical issues of human cloning

See also: Christian views on cloning

Roman Catholicism and many conservative Christian groups have opposed human cloning and the cloning of human embryos, believing that a human life begins the moment a human egg becomes fertilized. Other Christian denominations such as the United Church of Christ do not believe a fertilized egg constitutes a living being, but still they oppose the cloning of embryonic cells. The World Council of Churches, representing nearly 400 denominations worldwide, opposed cloning of both human embryos and whole humans in February 2006. The United Methodist Church opposed research and reproductive cloning in May 2000 and again in May 2004.

Libertarian views on the subject suggest that the federal government of the United States does not have the power to regulate cloning, as it is not given any such authority by the US constitution. (Similar to abortion rights.)

At present, the main objection to human cloning is that the cloned individual may be biologically damaged, due to the inherent unreliability of its origin: researchers currently are unable to safely and reliably clone non-human primates.

However, many believe that as cloning research and methods improve, concerns of safety and reliability will no longer be an issue. However, it must be pointed out that this has yet to occur, and may never occur. Rudolph Jaenisch, a professor at Harvard, has pointed out that we have become more efficient at producing clones which are still defective (Development Dynamics. Volume 235, pages 2460-2469. 2006). Other arguments against cloning come from various religious orders (believing cloning violates God's will or the natural order of life), and a general discomfort some have with the idea of "meddling" with the creation and basic function of life. This unease often manifests itself in contemporary novels, movies, and popular culture, as it did with numerous prior scientific discoveries and inventions. Various fictional scenarios portray clones being unhappy, soulless, or unable to integrate into society. Furthermore, clones are often depicted not as unique individuals but as "spare parts," providing organs for the clone's original (or any non-clone that requires replacement organs).

Needless to say, cloning is a poignant and important topic, reflected by its frequent discussion and debate among politicians, scientists, the media, religions, and the general public.

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.^[5]

In 2000, 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 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.^[6][7]

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.^[8] However, on 2005-02-15 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 2005-05-15, it was announced that the Thylacine project would be revived, with new participation from researchers in New South Wales and Victoria.

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 cloned, the question would remain open if they would be viable at all in the absence of parents that could teach or show them natural behavior. Essentially, if cloning an extinct species succeeded — it must be considered that cloning still is an experimental technology that succeeds only by chance — it is far 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 and environmentalists vehemently oppose cloning endangered species — not because they think it won't work but 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 concludes that cloning in animal conservation is an experimental technology that, at its present state, cannot be expected to work except by pure chance and utterly fails a cost-benefit analysis.^[9] Furthermore, he says, 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 concludes:

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.[9]

Embryo

Main article: Somatic cell nuclear transfer

Somatic cell nuclear transfer can also be used to create a clonal embryo. The most likely scenario for this is to produce embryos for use in research, particularly stem cell research. This process is also called "research cloning" or "therapeutic cloning."

Therapeutic cloning, also called "embryo cloning," is the production of human embryos for use in research. The goal of this process is not to create cloned human beings, but rather to harvest stem cells that can be used to study human development and to treat disease. Stem cells are important to biomedical researchers because they can be used to generate virtually any type of specialized cell in the human body. Stem cells are extracted from the egg after it has divided for 5 days. The egg at this stage of development is called a blastocyst. The extraction process destroys the embryo, which raises a variety of ethical concerns. Many researchers hope that one day stem cells can be used to serve as replacement cells to treat heart disease, Alzheimer's, cancer, and other diseases.

Scientists believe that cloning may be used to create stem cells genetically compatible with the somatic cell donor. Cloning in stem cell research, called research cloning or therapeutic cloning, has not yet been successful: no embryonic stem cell lines have been derived from clonal embryos. The process might provide a way to grow organs in host carrier, which become completely compatible with the original. Host carrier growing poses a risk of trans-species diseases if the host is of a different species (e.g., a pig).

In human beings, this is a highly controversial issue for several reasons. It involves creating human embryos in vitro and then destroying them, attempting to obtain embryonic stem cells. But proposals to use cloning techniques in human stem cell research raise a set of concerns beyond the moral status of the embryo. These have led a number of individuals and organizations who are not opposed to human embryonic stem cell research to be concerned about, or opposed to, human research cloning. One concern is that cloning in human stem cell research will lead to the reproductive cloning of humans. A second concern is the appropriate sourcing of the eggs that are needed. Research cloning requires a large number of human eggs, which can only be obtained from women. A third concern is the feasibility of developing stem cell therapies from cloning.

In November 2001, scientists from Advanced Cell Technologies (ACT), a biotechnology company in Massachusetts, announced that they had cloned the first human embryos for the purpose of advancing therapeutic research. To do this, they collected eggs from women's ovaries and then removed the genetic material from these eggs with a needle less than 2/10,000th of an inch wide. A skin cell was inserted inside the enucleated egg to serve as a new nucleus. The egg began to divide after it was stimulated with a chemical called ionomycin. The results were limited in success. Although this process was carried out with eight eggs, only three began dividing, and only one was able to divide into six cells before stopping.

References

External links and references

• 'Cloning' Freeview video by the Vega Science Trust and the BBC/OU
• Clone Guide - Cloning News Website with a Resource to Cloning information in the World
• The Reproductive Cloning Network. Cloning articles, resources and links
• Cloning in Focus, an accessible and comprehensive look at cloning research from the University of Utah's Genetic Science Learning Center
• Click and Clone. Try it yourself in the virtual mouse cloning laboratory, from the University of Utah's Genetic Science Learning Center
• Cloning timeline: from CNN
• "Cloning Addendum: A statement on the cloning report issues by the President's Council on Bioethics," The National Review, July 15, 2002 8:45am
• The President's Council on Bioethics
• Cloning educational resources and news from LiveScience.com