Much confusion happens when people see the word "clone" used. Depending on the age of the dictionary, the definition of biological cloning can be:
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.
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.
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.
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.
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.
See also Links for web sites relating to cloning and nuclear transfer.
Última modificación: Sábado, 11 de Junio de 2005