Велика бібліотека української літератури

Cloning

Cloning is the process of creating an identical copy of something. In , it collectively refers to processes used to create copies of fragments (Molecular Cloning), (Cell Cloning), or . The term also encompases situations, whereby organisms reproduce , but in common parlance refers to intentionally created copies of organisms.
Etymology
The term clone is derived from клщн, the word for "twig", referring to the process, whereby a new plant can be created from a twig. In , 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
Molecular cloning refers to the procedure of isolating a defined DNA sequence and obtaining multiple copies of it . Cloning is frequently employed to amplify DNA fragments containing , but it can be used to amplify any DNA sequence such as , 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 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 , a sequence element capable of directing the propagation of its self and any linked sequence. In practice, however, a number of other features are desired and a variety of specialised cloning exist that allow protein expression, tagging, single stranded 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 . 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 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 (б-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 .
Cellular cloning
Cloning a cell means to derive a (clonal) 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 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 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 or drug used to drive 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 rings (cloning rings), which have been dipped in grease are placed over an individual colony and a small amount of is added. Cloned cells are collected from inside the ring and transferred to a new vessel for further growth.
Organism
Organism cloning refers to the procedure of creating a new mutlicellular 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 ) and some insects.
Horticultural
The term clone is used in to mean all descendants of a single plant, produced by or . Many horticultural plant are clones, having been derived from a single individual, multiplied by some process other than sexual reproduction. As an example, some European cultivars of represent clones that have been propagated for over two millennia. Other examples are and . 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 scrutiny and is generally treated as an entirely different kind of operation.
Many , , , and other form . Parts of a large clonal colony often become detached from the parent, termed , to form separate individuals. Some plants also form asexually, termed , e.g. .
Animals
Clonal derivation exists in nature in some animal species and is referred to as . An example is the "Little " (), which is native to and but has spread throughout many tropical environments.
Therapeutic Cloning
Therapeutic Cloning refers to a procedure which allows the cloning of specific body parts and organs to be utilised for medical purposes. This has not yet being realised, but it is the subject of much active research. Currently, patients subjected to transplantation are administered immunosuppresant drugs to prevent recognition of the foreign transplant by their immune system and its subsequent rejection. The ability to clonally derive organs from the patients' own cells would abolish the need for immunosuppressant drugs and would allow the patients to live a life without the potentially serious side-effects of immunosuppresant drugs. More importantly, the ability to clonally derive organs would alleviate the current shortage of transplants and would possibly reduce waiting times for translants to become available.
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. was created by reproductive cloning technology. In a process called "" (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 in the of the enucleated egg. Mitochondria, which are 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 growing process.
Mutations also occur with every cell division so no two cells in an individual are identical. 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 death.
Species cloned
The modern cloning techniques involving have been successfully performed on several species. Landmark experiments in chronological order:
: (1952) Many scientists questioned whether cloning had actually occurred and unpublished experiments by other labs were not able to reproduce the reported results.
: (1963) In , cloned a fish. He published the findings in an obscure Chinese science journal which was never translated into English.
: (1996) From early embryonic cells by Steen Willadsen. Megan and Morag cloned from differentiated embryonic cells in June 1995 and in 1997.
: (female, January 2000) from embryo splitting
: (males, 2001) and (2005) Brazil
: "CC" (female, late 2001), , 2004, was the first cat cloned for commercial reasons
: , a john mule born -, was the first horse-family clone.
: , a Haflinger female born -, was the first horse clone.
For a complete list see: .
Health aspects
The success rate of cloning has been low: 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; 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 had accelerated aging. Aging of this type is thought to be due to the shortening of , regions at the tips of which prevent genetic threads from fraying every time a . 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. Dolly died in the year of . 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 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.
Human cloning
Human cloning is the creation of a identical copy of an existing, or previously existing , by growing cloned from that individual. The term is generally used to refer to artificial human cloning; human clones in the form of 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. 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 human organisms. 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. This practice is still morally questionable, but arguably less so than the process of harvesting said organs from a cloned human being.
The cloning described above is , not to be confused with in which only parts (such as an organ) are cloned using genetic material from a patient's tissues.
Ethical issues of human 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. Many religious groups oppose all forms of cloning, including the potentially life-saving cloning of individual organs, on the grounds that life begins at conception. Concerns also exist regarding the protection of the identity of the individual and the right to protect one's genetic identity. In addition, if technology eventually does allow for successful cloning of humans, prejudice may develop against clones, as if they were a "lesser" form of human being. Such prejudice could force clones into a kind of slavery or caste system. The possible social implications of an artificial human production scheme were famously explored in the novel .
References
Vogel, Gretchen (2000). "In Contrast to Dolly, Cloning Resets Telomere Clock in Cattle". Science 288: 641.
Pence, Gregory E. (1998). Who’s Afraid of Human Cloning?. Rowman & Littlefield.
Heidi B. Perlman. "Scientists Close on Extinct Cloning", Associated Press, 2000-10-08.
Pence, Gregory E. (2005). Cloning After Dolly: Who's Still Afraid?
Holloway, Grant. "Cloning to revive extinct species", CNN.com, 2002-05-28.
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