Cloning

Molecular cloning refers to the procedure whereby a DNA sequence is amplified by genetic engineering techniques. Cloning is frequently employed to amplify DNA fragments containing genes, an essential step in their subsequent analysis. Frequently, 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. In practice, localisation of the gene does not always enable one to amplify the relevant genomic sequence.

Cloning of any DNA sequence involves the following four steps: amplification, ligation, transfection, and screening/selection. Initially, the DNA fragment of interest needs to be amplified (many copies need to be produced). Amplification is commonly achieved by means of PCR. 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 that allow for ligation. The yield of the ligation is typically low and depends on the procedure employed. Following ligation the vector with the insert of interest is transfected to cells. Most commonly electroporation is employed, although a number of alternative techniques are available. Finally, the transfected cells are cultured. As the aforementioned procedures are of particularly low yield, there is the need to identify the cell colonies that have been transfected with the construct of interest containing the desired insertion sequence. Modern cloning vectors include selectable antibiotic resistance markers, which allow only for cells in which the vector has been transfected to grow. However this selection step does not guarantee that the DNA insert is present in the vector. Further investigation of the resulting colonies is required to confirm that cloning was successful. This can be accomplished by means of blue/white screening and/or PCR, possibly followed by DNA sequencing.

Cloning a cell means to derive a (clonal) population of cells from a single cell. This is an important in vitro procedure when the expansion of a single cell with certain characteristics is desired, for example in the production of gene-targeted ES cells. Most individuals began as a single cell and are therefore the result of clonal expansion in vivo.

Cloning means to create a new organism with the same genetic information as a cell from an existing one(identical). It is an asexual method of reproduction, where fertilization or inter-gamete contact does not take place. In a modern context, this can involve somatic cell nuclear transfer in which a cell of the organism to be cloned, with its nucleus containing the DNA, is transferred into an egg cell which has had its nucleus removed. As the nucleus contains almost all of the genetic information of a lifeform, the "host" egg cell will develop into an organism with genetically identical nuclear DNA to the nucleus "donor".

However, this process does not conserve the mitochondrial genome (of the nucleus donor) unless the nucleus and egg cell donor were from the same maternal lineage. Also mutations occur with every cell division so no two cells in an individual are identical, nor are clones. 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 inviability.

The first animal clone was a frog, cloned by Thomas J. King and Robert W. Briggs in 1952.

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, although the root systems may be genetically different for each grafted plant. Other important horticultural cloning methods are cuttings, tissue culture, layering and storage organs, such as bulbs, tubers and corms. These are 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 ethical scrutiny and is generally treated as an entirely different kind of operation.

Natural clones -- Cloning is very common in single-celled organisms, which clone themselves via binary fission, e.g. paramecium, bacteria, many algae. Cloning is also very widespread in plants, where it is often called vegetative reproduction. Vegetative reproduction in plants often involves the formation of adventitious roots or shoots.

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.

Cloning 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. In this species, circumstantial evidence from microsatellite DNA suggests that both queens and males may reproduce clonally in one population in Suriname. Some animals can clone themselves via fragmentation, e.g. starfish.

Ethical issues of cloning -- See also: Christian views on cloningRoman Catholicism and many conservative Christian groups have opposed human cloning and the cloning of human embryos, believing that a human life begins the moment a human egg becomes fertilized. Other Christian denominations such as the United Church of Christ do not believe a fertilized egg constitutes a living being, but still they oppose the cloning of embryonic cells. The World Council of Churches, representing nearly 400 denominations worldwide, opposed cloning of both human embryos and whole humans in February 2006. The United Methodist Church opposed research and reproductive cloning in May 2000 and again in May 2004.

Libertarian views on the subject suggest that it is within a person's constitutional rights to conduct this process, similar to abortion rights.

At present, the main objection to human cloning is that the cloned individual may be biologically damaged, due to the inherent unreliability of its origin: researchers currently are unable to safely and reliably clone non-human primates.

However, as cloning research and methods improve, concerns of safety and reliability will no longer be an issue. Other arguments against cloning come from various religious orders (believing cloning violate's God's will or the natural order of life), and a general discomfort some have with the idea of "meddling" with the creation and basic function of life. This unease often manifests itself in contemporary novels, movies, and popular culture, much like numerous other scientific discoveries and inventions before. Various fictional scenarios portray clones being unhappy, soulless, or unable to integrate into society.

Furthermore, clones are often depicted not as unique individuals but as "spare parts," providing organs for the clone's original (or any non-clone that requires replacement organs). Needless to say, cloning is a poignant and important topic, reflected by its frequent discussion and debate among politicians, scientists, the media, religions, and the general public.

Health aspects -- The success rate of cloning has been low: Dolly the sheep was born after 277 eggs were to create 29 embryos, which only produced three lambs at birth, only one of which lived, Dolly. 70 calves have been created from 9,000 attempts and one third of them died young; Prometea 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 Dolly the Sheep had accelerated aging. Aging of this type is thought to be due to shortening of telomeres, regions at the tips of chromosomes which prevent genetic threads 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 telomere were longer than normal. Dolly died in the year of 2003. Ian Wilmut said that Dolly's early death had nothing to do with cloning but with a respiratory infection common to lambs raised inside like Dolly.

Consistent with Dolly's telomeres being longer, analysis of the telomeres from cloned cows showed they 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 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 (1996-2003-), a 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 6. Her birth was announced on 1997-02-22.The name "Dolly" came from a suggestion by Jesse Haase who helped with her birth, in honor of Dolly Parton, because it was a mammary cell that was cloned. The technique that was made famous by her birth is somatic cell nuclear transfer, in which a non-reproductive cell containing a nucleus is placed in a de-nucleated ovum (which then develops into a fetus). When Dolly was cloned in 1996 from a cell taken from a six-year-old ewe, she became the center of much controversy that still exists today.

On 2003-04-09 her stuffed remains were placed at Edinburgh's Royal Museum, part of the National Museums of Scotland.Ian Wilmut's role in cloning Dolly the sheep is in doubt. In 2006 he admitted under oath in a Scottish court that he did not clone Dolly the Sheep and was not responsible for the scientific breakthrough which made it all possible. He credited somebody called Keith Campbell as being the brains behind Dolly the Sheep.

Therapeutic cloning is the procedure for creating stem cells genetically compatible with the patient. Therapeutic cloning might provide a way to grow organs in host carrier, which become completely compatible with the original. 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, as it involves creating human embryos in vitro and then destroying them to obtain multipotent embryonic stem cells.

Human cloning is the creation of a genetically identical copy of an existing, or previously existing human or growing cloned tissue from that individual. 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.

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 2000, 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 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 endagnered 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.