Technical Aspects of Cloning
What is cloning?
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.
Method of nuclear transfer to produce Dolly
The technique involves several steps (see diagram). 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. This was the first time live sheep 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.
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.