Cloning

For the cloning of human beings, see human cloning. For other uses, see clone.

Overview

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 "twig, branch", 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

Main article: 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 vectors 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.Do not delete my edits or ill delete the deleters page!!!

Cellular cloning

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 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

Main article: 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 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, 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

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.

First cloned buffalo

On September 15, 2007, the Philippines announced its development of Southeast Asia’s first cloned water buffalo. The Philippine Council for Agriculture, Forestry and Natural Resources Research and Development (PCARRD), under the Department of Science and Technology in Los Baños, Laguna appoved this project. The Department of Agriculture’s Philippine Carabao Center (PCC) will implement “Cloning through somatic cell nuclear transfer as a tool for genetic improvement in water buffaloes.” “Super buffalo calves” will be produced. There will be no modification or alteration of the genetic materials, as in GMOs (genetically modified organisms).

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 bladders, early cleavage-stage human embryos have been cloned 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:

• 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.
• 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
• 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.

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 telomeres, regions at the tips of chromosomes 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. 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.

Analysis of telomeres from cloned cows showed that they were longer than noncloned calves. 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 aging 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.

Dolly the Sheep

Main article: 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

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 of identical 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. A third type of cloning called replacement cloning exists in theory, and is a combination of therapeutic and reproductive cloning. Replacement cloning entails the replacement of an extensively damaged, failed, or failing body through cloning followed by whole or partial brain transplant. It has been proposed as a way to greatly extend lifespan.

Human cloning is among the most controversial forms of the practice. There have been numerous demands for all progress in the human cloning field to be halted. Some people and groups oppose therapeutic cloning but many more 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 . Serious ethical issues have arisen in discussions of harvesting of organs from clones. 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.

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.

In 2001, 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.

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. 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 their 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. 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.

”

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. 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 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.

External links and references

• Cloning Fact Sheet from Human Genome Project Information website.
• '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

References

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