Factsheet on
Cloning
Courtesy of the Roslin Institute
"Clone"
Defined
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.
Technology of
cloning
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.
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.
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