Technology

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.

In 1996, Roslin Institute and collaborators PPL Therapeutics created Dolly, the first animal cloned from a cell taken from an adult animal. The announcement of her birth in February 1997 started the current fascination in all things cloned. Until then, almost all biologists thought that the cells in our bodies were fixed in their roles: the creation of Dolly from a mammary gland cell of a six year old sheep showed this was not the case and the achievement was voted Science Breakthrough of the Year at the end of 1997.

progress AD (After Dolly)

At first Dolly was a 'clone alone' but in August 1998, a group in Hawaii published a report of the cloning of over 50 mice by nuclear transfer. Since then, research groups around the world have reported the cloning of cattle, sheep, mice, goats and pigs. Equally competent groups have had no success in cloning rabbits, rats, monkeys, cats or dogs.

There are differences in early development between species that might influence success rate. In sheep and humans, the embryo divides to between the 8- and 16- cell stage before nuclear genes take control of development, but in mice this transition occurs at the 2 cell stage. In 1998, a Korean group claimed that they had cloned a human embryo by nuclear transfer but their experiment was terminated at the 4-cell stage and so they had no evidence of successful reprogramming.

Success rates remain low in all species, with published data showing that on average only about 1% of 'reconstructed embryos' leading to live births. With unsuccessful attempts at cloning unlikely to be published, the actual success rate will be substantially lower. Many cloned offspring die late in pregnancy or soon after birth, often through respiratory or cardiovascular dysfunction. Abnormal development of the placenta is common and this is probably the major cause of foetal loss earlier in pregnancy. Many of the cloned cattle and sheep that are born are much larger than normal and apparently normal clones may have some unrecognised abnormalities.

The high incidence of abnormalities is not surprising. Normal development of an embryo is dependent on the methylation state of the DNA contributed by the sperm and egg. and on the appropriate reconfiguration of the chromatin structure after fertilisation. Somatic cells have very different chromatin structure to sperm and 'reprogramming' of the transferred nuclei must occur within a few hours of activation of reconstructed embryos. Incomplete or inappropriate reprogramming will lead to dysregulation of gene expression and failure of the embryo or foetus to develop normally or to non-fatal developmental abnormalities in those that survive.

Improving success rates is not going to be easy. At present, the only way to assess the 'quality' of embryos is to look at them under the microscope and it is clear that the large majority of embryos that are classified as 'normal' do not develop properly after they have been implanted. A substantial effort is now being made to identify systematic ways of improving reprogramming. One focus is on known mechanisms involved in early development, and in particular on the 'imprinting' of genes. Another is to use technological advances in genomics to screen the expression patterns of tens of thousands of genes to identify differences between the development of 'reconstructed embryos' and those produced by in vivo or in vitro fertilisation.

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.

Other obvious requirements for cloning are an appropriate supply of oocytes and surrogate mothers to carry the cloned embryos to term. Cloning of endangered breeds will be possible by using eggs and surrogates from more common breeds of the same species. It may be possible to clone using a closely related species but the chance of successfully carrying a pregnancy to term would be increasingly unlikely if eggs and surrogate mothers are from more distantly related species. Proposals to 'save' the Panda by cloning, for example, would seem to have little or no chance of success because it has no close relatives to supply eggs or carry the cloned embryos.

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.

Ethics

Many ethical and moral concerns have arisen over the potential applications of the cloning technology. The technology is still in its infancy and in the meantime, society as a whole has time to contemplate which uses of the technology might be acceptable and which would not. The suddenness of the news of the cloning of the first adult animal caught almost all commentators by surprise and some suggested that we should have fully discussed the implications of our work before we started. The public may see science as a series of 'breakthroughs' but in reality progress is much more continuous. Where in the sequence of events that led to Dolly should we have consulted and with whom? It is also impossible to predict all potential applications of a new technology. Most will be beneficial but all technology can be misused in one way or another. The solution is not to regulate the technology itself but how it is applied.

Those concerned that scientists were "playing at God" seemed to ignore how much mankind has altered the cards that we were originally dealt. Animals were first domesticated about 5000 years ago and selective breeding since has produced modern strains of livestock, plants and pets which are very different from their original progenitors. In medicine, our current life expectancy of well over 70 years is a result of direct intervention in nature, from improved prenatal care, vaccination and use of antibiotics. The human condition is still far from perfect and there is no particular reason now to call a general halt to what most people view as progress.

Roslin believes it has a clear social responsibility to keep the public informed of the results of its research and is a very active participant in the ongoing public debates about cloning, animal experimentation, genetic modification and human stem cell research.

(source:www.prodiversitas.bioetica.org/clonacion2.htm#technology)