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Cloning is the process of creating an identical copy of something. In Biology , it collectively refers to processes used to create copies of DNA fragments (molecular cloning), Cells (cell cloning), or Organisms . The term also encompasses situations whereby organisms reproduce Asexually . ETYMOLOGY The term ''clone'' is derived from ''κλών'', the Greek word for "twin", 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 See Also: Molecular cloning Molecular cloning refers to the procedure of isolating a defined DNA sequence and obtaining multiple copies of it '' In Vivo ''. Cloning is frequently employed to amplify DNA fragments containing Genes , but it can be used to amplify any DNA sequence such as Promoters , non-coding sequences and randomly fragmented DNA. It is utilised in a wide array of biological experiments and practical applications such as 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. In essence, in order to amplify any DNA sequence in a living organism, that sequence must be linked to an Origin Of Replication , a sequence element capable of directing the propagation of itself and any linked sequence. In practice, however, a number of other features are desired and a variety of specialised Cloning Vector s 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, ligation, transfection, and screening/selection. Although these steps are invariable among cloning procedures a number of alternative routes can be selected, these are summarised as a ‘cloning strategy’. Initially, the DNA of interest needs to be isolated to provide a relevant DNA segment of suitable size. 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. A number of alternative techniques are available, such as chemical sensitivation of cells, electroporation 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 (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 is required to confirm that cloning was successful. This may be accomplished by means of PCR, restriction fragment analysis and/or DNA Sequencing . CELLULAR CLONING 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 higher 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 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. ORGANISM See Also: Asexual reproduction Organism 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. 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 Cultivar s are clones, having been derived from a single individual, multiplied by some process other than sexual reproduction. As an example, some European cultivars of Grape s 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 Tree s, Shrub s, Vine s, Fern s and other Herbaceous Perennial s 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 Seed s asexually, termed apomixis, e.g. Dandelion . Animals Clonal derivation 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. Therapeutic cloning Therapeutic cloning refers to a procedure which produces cells, specific body parts, and organs to be utilized for medical purposes. Although this has only been realized with parts of bladdershttp://www.usatoday.com/tech/columnist/aprilholladay/2006-05-15-cloning_x.htm, early cleavage-stage human embryos have been clonedhttp://sciam.com/print_version.cfm?articleID=0008B8F9-AC62-1C75-9B81809EC588EF21http://www.wired.com/wired/archive/12.01/clones.html and this is the subject of much active research. Currently, patients subjected to transplantation are administered Immunosuppressive Drugs to prevent recognition of the foreign transplant by their immune system and its subsequent rejection. The ability to clonally derive tissues and organs from the patients' own cells would abolish the need for immunosuppressive drugs and would allow the patients to live a life without the potentially serious side-effects of immunosuppressive drugs. More importantly, the ability to clonally derive organs would alleviate the current shortage of transplants and would possibly reduce waiting times for Transplants to become available. Reproductive cloning Reproductive cloning 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 which 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. Species cloned The modern cloning techniques involving Nuclear Transfer have been successfully performed on several species. Landmark experiments in chronological order:
For a complete list see: List Of Animals That Have Been Cloned . 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. 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 accelerated aging. Aging of this type is thought to be due to the shortening of Telomere s, regions at the tips of Chromosome s which prevent genetic threads from 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 telomeres were longer than normal. Dolly died in the year 2003 . Ian Wilmut said that Dolly's early death had nothing to do with cloning but with a respiratory infection common to lambs raised 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 the effect 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.1 Dolly the Sheep See Also: Dolly the Sheep Dolly ( 1996-07-05 – 2003-02-14 ), a Finn Dorsett Ewe , was the first mammal to have been successfully cloned from an adult cell. She was cloned at the Roslin Institute in Scotland and lived there until her death when she was six. On 2003-04-09 her Stuffed Remains were placed at Edinburgh's Royal Museum , part of the National Museums Of Scotland . Dolly was publically significant because the effort showed that the genetic material from a specific adult cell, programmed to express only a distinct subset of its genes, could be reprogrammed to grow an entire new organism. Before this demonstration, there was no proof for the widely spread hypothesis that differentiated animal cells can give rise to entire new organisms. Human cloning See Also: Human cloning Human cloning is the creation of a . It has been proposed as a way to greatly extend lifespan. Embryo See Also: 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 Disease s. 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 carriers, so that organs could be produced which would be completely compatible with the original tissue donor. 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 during the process of 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 in principle 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 relates to 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. REFERENCES EXTERNAL LINKS AND REFERENCES
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