Cloning in biology is the process of producing similar populations of genetically identical individuals that occurs in nature when organisms

such asbacteria, 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 (kln, twig), referring to the process whereby a new plant can be created from a twig. Inhorticulture, 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". [1][2]Since the term entered the popular lexicon in a more general context, the spelling clone has been used exclusively. In botany, the term lusus was traditionally used.[3] 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. Such practice has met strong resistance in other regions, such as Europe, particularly over the labeling issue.
Contents
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1 Molecular cloning 2 Cellular cloning

o o

2.1 Unicellular organisms 2.2 Cloning in stem cell research

3 Organism cloning

o o o

3.1 Horticultural 3.2 Parthenogenesis 3.3 Artificial cloning of organisms

3.3.1 First Moves 3.3.2 Methods

3.3.2.1 Obtaining Blastocysts

3.3.3 Dolly the Sheep 3.3.4 Water buffalo 3.3.5 Species cloned 3.3.6 Human cloning 3.3.7 Ethical issues of cloning 3.3.8 Cloning extinct and endangered species

4 In science fiction 5 See also 6 References 7 External links

[edit]Molecular

cloning

Main article: Molecular cloning

Molecular cloning refers to the process of making multiple molecules. Cloning is commonly used to amplify DNA fragments containing whole genes, but it can also be used to amplify any DNA sequence such as promoters, non-coding sequences and randomly fragmented DNA. It is used in a wide array of biological experiments and practical applications ranging from genetic fingerprinting to large scale protein production. Occasionally, the term cloning is misleadingly used to refer to the identification of the chromosomal location of a gene associated with a particular phenotype of interest, such as in positional cloning. In practice, localization of the gene to a chromosome or genomic region does not necessarily enable one to isolate or amplify the relevant genomic sequence. To amplify any DNA sequence in a living organism, that sequence must be linked to an origin of replication, which is a sequence of DNA capable of directing the propagation of itself and any linked sequence. However, a number of other features are needed and a variety of specialised cloning vectors (small piece of DNA into which a foreign DNA fragment can be inserted) exist that allow protein expression, tagging, single stranded RNA and DNA production and a host of other manipulations. Cloning of any DNA fragment essentially involves four steps[4] 1. 2. 3. 4. 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. [edit]Cellular

cloning
organisms

[edit]Unicellular

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).[5] 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. [edit]Cloning

in stem cell research

Main article: Somatic cell nuclear transfer 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. [6] 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. \ The oocyte will react on the somatic cell nucleus, the same way it would on sperm cells.[9]

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.[9] 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. 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.[9]

[edit]Organism

cloning

Further information: Asexual reproduction 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 intergamete contact does not take place. Asexual reproduction is a naturally occurring phenomenon in many species, including most plants (seevegetative 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,[11] has been common practice in the horticultural world for hundreds of years. [edit]Horticultural The term clone is used in horticulture to refer to descendants of a single plant which were produced by vegetative reproduction or apomixis. Many horticultural plant cultivars are clones, having been derived from a single individual, multiplied by some process other than sexual reproduction. As an example, some European cultivars of grapes represent clones that have been propagated for over two millennia. [edit]Parthenogenesis Clonal derivation exists in nature in some animal species and is referred to as . 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. . [edit]Methods Reproductive cloning generally uses "somatic cell nuclear transfer" (SCNT) to create animals that are genetically identical. This process entails the transfer of a nucleus from a donor adult cell (somatic cell) to an egg 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 mitochondrial DNA is wholly from the cytoplasmic donor's egg, thus the mitochondrial genome is not the

same as that of the nucleus donor cell from which it was produced. This may have important implications for cross-species nuclear transfer in which nuclear-mitochondrial incompatibilities may lead to death. [edit]Obtaining Blastocysts 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. [14] [edit]Species cloned

Tadpole: (1952) Robert Briggs and Thomas J. King had successfully cloned northern leopard frogs: thirty-five complete embryos and twenty-seven tadpoles from one-hundred and four successful nuclear transfers.[19], but many scientists questioned whether cloning had actually occurred and unpublished experiments by other labs were not able to reproduce the reported results.[citation needed]

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.[clarification needed][21] Sheep: (1996) From early embryonic cells by Steen Willadsen. Megan and Morag[22] cloned from differentiated embryonic cells in June 1995 and Dolly the sheep from a somatic cell in 1997.[23] Rhesus Monkey: Tetra (January 2000) from embryo splitting[24][clarification needed][25] Gaur: (2001) was the first endangered species cloned.[26] Cattle: Alpha and Beta (males, 2001) and (2005) Brazil[27] Cat: CopyCat "CC" (female, late 2001), Little Nicky, 2004, was the first cat cloned for commercial reasons [28] Dog: Snuppy, a male Afghan hound was the first cloned dog (2005).[29] Rat: Ralph, the first cloned rat (2003)[30] Mule: Idaho Gem, a john mule born 4 May 2003, was the first horse-family clone.[31] Horse: Prometea, a Haflinger female born 28 May 2003, was the first horse clone. [32]

[edit]Human cloning 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 ofidentical 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.[37] 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 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.[51] 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. [51] 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.[7] Results of these abnormally expressed genes in the cloned mice were premature death, pneumonia, liver failure and obesity.[7]