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Cloning is the process of producing genetically identical individuals of an organism either naturally or artificially. In nature, many organisms produce clones through asexual reproduction. Cloning in biotechnology refers to the process of creating clones of organisms or copies of cells or DNA fragments. Beyond biology, the term refers to the production of multiple copies of digital media or software. The term clone, coined by Herbert J. Webber, is derived from the Ancient Greek word º»Î½ klMn, ‘twig’, referring to the process whereby a new plant can be created from a twig. In botany, the term lusus was traditionally used. In horticulture, the spelling clon was used until the twentieth century; the final e came into use.
Natural cloning is a natural form of reproduction that has allowed life forms to spread for hundreds of millions of years. It is the reproduction method used by plants, fungi, and bacteria, and is also the way that clonal colonies reproduce themselves. Examples of these organisms include blueberry plants, hazel trees, the Pando trees, the Kentucky coffee tree, Myrica, and the American sweetgum.
DNA
Although these steps are invariable among cloning procedures several 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. The vector 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. Several alternative techniques are available, such as chemical sensitization 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 color selection markers, which provide blue/white screening on an X-gal medium. Nevertheless, these selection steps do not 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 using PCR, restriction fragment analysis, and/or DNA sequencing.
Cloning organisms
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. In 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 cells, sterile polystyrene 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 stem cells
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, but rather to harvest stem cells that can be used to study human development and 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 removing the nucleus from an egg cell and inserting a nucleus from the adult cell to be cloned. 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 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. As the procedure could not be automated and had to be performed manually under a microscope, SCNT was very resource-intensive. The biochemistry involved in reprogramming the differentiated somatic cell nucleus and activating the recipient egg was also far from being well understood. However, by 2014 researchers were reporting cloning success rates of seven to eight out of ten, and in 2016, a Korean Company Sooam Biotech was reported to be producing 500 cloned embryos per day.
In SCNT, not all of the donor cell’s genetic information is transferred, as the donor cell’s mitochondria that contain their mitochondrial DNA are left behind. The resulting hybrid cells retain those mitochondrial structures that 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
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 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 a common practice in the horticultural world for hundreds of years.
Dolly the sheep
Dolly, a Finn-Dorset ewe, was the first mammal to have been successfully cloned from an adult somatic cell. Dolly was formed by taking a cell from the udder of her 6-year-old biological mother. Dolly’s embryo was created by taking the cell and inserting it into a sheep’s ovum. It took 434 attempts before an embryo was successful. The embryo was then placed inside a female sheep that went through a normal pregnancy. She was cloned at the Roslin Institute in Scotland by British scientists Sir Ian Wilmut and Keith Campbell and lived there from her birth in 1996 until she died in 2003 when she was six. She was born on 5 July 1996 but was not announced to the world until 22 February 1997. 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 genetic material from a specific adult cell, designed to express only a distinct subset of its genes, can be redesigned 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 has not yet been demonstrated in a mammalian system.
The first mammalian cloning had a success rate of 29 embryos per 277 fertilized eggs, which produced three lambs at birth, one of which lived. In a bovine experiment involving 70 cloned calves, one-third of the calves died quite young. The first successfully cloned horse, Prometea, took 814 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 problems with the cloning process. This idea that the nuclei have not irreversibly aged was shown in 2013 to be true for mice.
Ethical issues of cloning
There are a variety of ethical positions regarding the possibilities of cloning, especially human cloning. While many of these views are religious in origin, the questions raised by cloning are faced by secular perspectives as well. Perspectives on human cloning are theoretical, as human therapeutic and reproductive cloning are not commercially used; animals are currently cloned in laboratories and livestock production.
Advocates support the development of therapeutic cloning to generate tissues and whole organs to treat patients who otherwise cannot obtain transplants, to avoid the need for immunosuppressive drugs, Advocates for reproductive cloning believe that parents who cannot otherwise procreate should have access to the technology.
Opponents of cloning have concerns that technology is not yet developed enough to be safe and that it could be prone to abuse, as well as concerns about how cloned individuals could integrate with families and with society at large.
Religious groups are divided, with some opposing the technology as usurping ‘God’s place’ and, to the extent embryos are used, destroying a human life; others support therapeutic cloning’s potential life-saving benefits.
Cloning of animals is opposed by animal groups due to the number of cloned animals that suffer from malformations before they die, and while food from cloned animals has been approved by the US FDA, its use is opposed by groups concerned about food safety.
Cloning extinct and endangered species
Cloning, or more precisely, the reconstruction of functional DNA from extinct species has, for decades, been a dream. Possible implications of this were dramatized in the 1984 novel Carnosaur and the 1990 novel Jurassic Park. The best current cloning techniques have an average success rate of 9.4 percent and might be used to clone extinct species. Anticipating this possibility, tissue samples from the last bucardo 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.
In 2002, geneticists at the Australian Museum announced that they had replicated the DNA of the thylacine, at the time extinct for about 65 years, using a polymerase chain reaction. However, on 15 February 2005, the museum announced that it was stopping the project after tests showed the specimens’ DNA had been too badly degraded by the 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 2003, 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 frozen cell nucleus 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 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 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 would 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. It was noted, however, that the result, if possible, would be an elephant-mammoth hybrid rather than a true mammoth. Another problem is the survival of the reconstructed mammoth: ruminants rely on a symbiosis with specific microbiota in their stomachs for digestion.
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