Much confusion happens when people see the word "clone" used. Depending on the age of the dictionary, the definition of biological cloning can be:

• A group of genetically identical individuals descended from the same parent by asexual reproduction. Many plants show this by producing suckers, tubers or bulbs to colonise the area around the parent.
• A group of genetically identical cells produced by mitotic division from an original cell. This is where the cell creates anew set of chromosomes and splits into two daughter cells. This is how replacement cells are produced in your body when the old ones wear out.
• A group of DNA molecules produced from an original length of DNA sequences produced by a bacterium or a virus using molecular biology techniques. This is what is often called molecular cloning or DNA cloning
• The production of genetically identical animals by 'embryo splitting'. This can occur naturally at the two cell stage to give identical twins. In cattle, when individual cells from 4- and 8-cell embryos and implanted in different foster mothers, they can develop normally into calves and this technique has been used routinely within cattle breeding schemes for over 10 years.
• The creation of one or more genetically identical animals by transferring the nucleus of a body cell into an egg from which the nucleus has been removed. This is also known as Nuclear Transfer (NT) or cell nuclear replacement (CNR) and is how Dolly was produced.

Nuclear transfer involves transferring the nucleus from a diploid cell ( containing 30-40,000 genes and a full set of paired chromosomes) to an unfertilised egg cell from which the maternal nucleus has been removed. The technique involves several steps (see diagram below). The nucleus itself can be transferred or the intact cell can be injected into the oocyte. In the latter case, the oocyte and donor cell are normally fused and the 'reconstructed embryo' activated by a short electrical pulse. In sheep, the embryos are then cultured for 5-6 days and those that appear to be developing normally ( usually about 10%) are implanted into foster mothers.

Nuclear transfer is not a new technique. It was first used in 1952 to study early development in frogs and in the 1980's the technique was used to clone cattle and sheep using cells taken directly from early embryos. In 1995, Ian Wilmut, Keith Campbell and colleagues created live lambs- Megan and Morag - from embryo derived cells that had been cultured in the laboratory for several weeks. This was the first time live animals had been derived from cultured cells and their success opened up the possibility of introducing much more precise genetic modifications into farm animals.

Limitations of nuclear transfer

It is important to recognise the limitations of nuclear transfer. Plans to clone extinct species have attracted a lot of publicity. One Australian project aims to resurrect the 'Tasmanian tiger' by cloning from a specimen that had been preserved in a bottle of alcohol for 153 years and another research group announced plans to clone a mammoth from 20,000 year old tissue found in the Siberian permafrost. However, the DNA in such samples is hopelessly fragmented and there is no chance of reconstructing a complete genome. In any case, nuclear transfer requires an intact nucleus, with functioning chromosomes. DNA on its own is not enough: many forget that Jurrasic Park was a work of fiction.

Method of nuclear transfer in livestock

Applications

Nuclear transfer can viewed in two ways: as a means to create identical copies of animals or as a means of converting cells in culture to live animals. the former has applications in livestock production, the latter provides for the first time an ability to introduce precise genetic modifications into farm animal species.

• Cloning in Farm Animal production
  Nuclear transfer can in principle be used to create an infinite number of clones of the very best farm animals. In practice, cloning would be limited to cattle and pigs because it is only in these species that the benefits might justify the costs. Cloned elite cows have already been sold at auction for over $40,000 each in the US but these prices reflect their novelty value rather than their economic worth. To be effective, cloning would have to be integrated systematically into breeding programmes and care would be needed to preserve genetic diversity. It would also remains to be shown that clones do consistently deliver the expected commercial performance and are healthy and that the technology can be applied without compromising animal welfare ( see Farm Animal Welfare Council Report).

• Production of Human therapeutic proteins
  Human proteins are in great demand for the treatment of a variety of diseases. Whereas some can be purified from blood, this is expensive and runs the risk of contamination by AIDS or hepatitis C. Proteins can be produced in human cell culture but costs are very high and output small. Much larger quantities can be produced in bacteria or yeast but the proteins produced can be difficult to purify and they lack the appropriate post-translational modifications that are needed for efficacy in vivo.

  By contrast, human proteins that have appropriate post-translational modifications can be produced in the milk of transgenic sheep, goats and cattle. Output can be as high as 40 g per litre of milk and costs are relatively low. PPL Therapeutics, one of the leaders in this field and their lead product, alpha-1-antitrypsin, is due to enter phase 3 clinical trials for treatment of cystic fibrosis and emphysema in 2001.. Nuclear transfer allows human genes to be inserted at specific points in the genome, improving the reliability of their expression and allows genes to be deleted or substitutes as well as added.

• Xenotransplantation
  The chronic shortage of organs means that only a fraction of patients who could benefit actually receive transplants. Genetically modified pigs are being develop as an alternative source of organs by a number of companies, though so far the modifications have been limited to adding genes. Nuclear transfer will allow genes to be deleted from pigs and much attention is being directed to eliminating the alpha-galactosyl transferase gene. This codes for an enzyme that creates carbohydrate groups which are attached to pig tissues and which would be largely responsible for the immediate rejection of an organ from a normal pig by a human patient.
  

• Cell Based Therapies
  Cell transplants are being developed for a wide variety of common diseases, including Parkinson's Diseases, heart attack, stroke and diabetes. Transplanted cells are as likely to be rejected as organs but this problem could be avoided if the type of cells needed could be derived from the patients themselves. The cloning of adult animals from a variety of cell types shows that the egg and early embryo have the capability of 'reprogramming' even fully differentiated cells. Understanding more about the mechanisms involved may allow us to find alternative approaches to 'reprogramming' a patient's own cells without creating ( and destroying ) human embryos.