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Dolly the sheep, the world's first cloned mammal, so far she has been very happy. She got married and gave birth to her babies. Everything was fine.
However now the scientist reported that she has slight DNA damage problem. This latest information could have an impact on how cloning technology will be used in the future.
Dolly and two other cloned sheep have shorter telomeres -- the tiny strands of DNA at the end of chromosomes that scientists believe may hold the secrets to youth and aging.
Telomeres shorten each time a cell divides and are progressively eroded as an animal ages. As the telomeres shorten the chromosomes can become unstable and prone to damage.
Researchers from Edinburgh's Roslin Institute and PPL Therapeutics said Dolly, who was cloned from a cell taken from a six-year-old sheep, had telomeres that were about 20 percent shorter than those of other sheep of a similar age.
``We don't see any signs of any problem in Dolly and the other animals but perhaps it is too early to note such a problem. We just don't know,'' PPL's Alan Colman said.
The finding, reported in a letter to "Nature", is not unexpected. Scientists know that cell culture leads to a shortening of telomeres and cells used for Dolly and the other sheep were cultured in the laboratory. In Dolly's case the shortening was compounded because she was derived from a cell from a middle-aged animal.
``It reminds people this is an issue that should be called into account in any of the proposals for, not so much agricultural cloning, but some of the uses in human therapeutic cloning where extensive time periods of culture in vitro would be necessary. It could be an issue,'' Colman said.
Peter Gladstone
Superstar storm
Dolly is a cloning superstar, the world's most photographed sheep. She is
quite happy to perform in front of the cameras, and soon will find her boyfriend . . . The
birth of Dolly had stormed the whole human society. It inspired so much concern, worry,
excitement, agitation and debates in human beings, why?
Animal cloning experiments have been carried out for many years, and it
is well known that nuclei taken from early embryos or embryo-drived cells of animals can
be used to produce adult clones.
There were many examples, for instance, in March 1996, the same Wilmut's
group in Roslin Institute reported the successful cloning of sheep from embryo-derived
cells. Those cells had been cultured in the laboratory for 6 - 13 passages.
After Dolly's report in February 97, the Oregon Regional Primate Research
Center claimed that they have cloned two Rhesus monkeys from early stage embryos.
And in August 97, ABS Global announced their cloned calf, called Gene. It
was cloned by using primordial stem cells from a 30-day-old calf fetus.
However Dolly has been the only superstar, majorly because she was cloned
by an adult cell (or as we called it "sometic cell").
What is the difference? As far as the worries are concerned: If this
technique is misapplied to human cloning, at least a large number of human embryos are not
so easy to obtain . . . but the adult cells - it can be got from any tiny part of the
human body, the blood, the skin, the hair etc. It will be impossible to ban all the
sources. In addition, the possibility of cloning past famous persons from their remains
will be a terrible dream . . .
From the view of scientist, certain basic questions have to be addressed -
for instance, how could the adult cell nucleus be reprogrammed or remodeled to an
embryonic state?
How Dolly was produced
In this article, I would briefly provide the scientific fact, how could an
adult animal be cloned by an adult cell. To show the cloning process step by step, it is
not so mysterious or frightening as one imagines.
The Procedure of Cloning Dolly
Step 1: The Donor-Cell population
Wilmut and his group took cells from 3 different tissues of ewes.
(a) From a day-9 embryo of a Poll Dorset ewe - embryo disc cells. (Passage
7 - 9 cells were used as nuclear donors.)
(b) From a day-26 fetus of a Black Welsh ewe - fetal fibroblast cells.
(Passage 4 - 6 cells were used as nuclear donors.)
(c) From mammary gland of a 6-year old Finn Dorset ewe - mammary
epithelium cells. (Passage 3 - 6 cells were used as nuclear donors.)
Totally they did 834 nuclear transfers and 8 live lambs were born from the cloning experiment. (Four cloned lambs out of 385 fusions by the donor embryo cells. Three cloned lambs out of 172 fusions by the fetus cells. And a single Dolly from the 277 fusions of the adult cells. )
Now we would like to concentrate on Dolly's cloning.
Step 2: Inducing donor cells to become quiescent
The primary cell culture from the mammary gland of a 6-year-old Finn
Dorset ewe were a mixture of mammary epithelial cells (>90%), myoepithelial cells and
fobroblasts. The possibility of a small proportion of undifferentiated stem cells could
not be excluded.
An important step was to induce these donor cells to exit the growth
cycle and enter the G0 phase of the cell cycle before nuclear transfer.
This was simply accomplshed by starving these cells - reducing the
concentration of serum in the culture medium from 10% to 0.5% for 5 days.
Step 3: Oocytes
Oocytes were obtained from Scottish Blackface ewes between 28 - 33 hours
after injection of gonadotropin-releasing hormone (GnRH) and enucleated as soon as
possible.
Enucleation method: A small amount of cytoplasm enclosed in pl;asma
membrane was removed from directly beneath the 1st polar body using a glass pipette (~20um
tip external diameter). Enucleation was confirmed by exposing this karyoplast to UV light
and checking for the presence of a metaphase plate.
Enucleated oocytes were recovered in calcium- and magnesium-free PBS
containing 1% fetal calf serum and transferred to calcium-free M2 medium containing 10%
fetal calf serum at 37oC.
Step 4: Nuclear transfer - fusion
Fusion of the donor cell to the enucleated oocyte and activation of the
oocyte were induced by the electrical pulses in 0.3M mannitol, 0.1mM MgSO4, 0.0005mM CaCl2.
For activation a single DC pulse of 1.25kV cm-1 for 80 us, and for fusion
an AC pulse of 3V for 5s followed by 3 DC pulses of 1.25 kV cm-1 for 80 us were applied.
Fusions were induced between 34 - 36 hours after GnRH injection to donor
ewes.
Step 5: Embryo -Morula/Blastocyst - Pregnancy Recipient
The fused couplets were cultured in ligated oviducts of sheep. After 6
days of culture, most reconstructed embryos were developed to morula or blastocyst.
One, two or three embryos were transferred to each recipient ewes and
allowed to develop to term.
In Dolly's group, 29 morula/blastocysts were
transferred to 13 recipients. Ultrasound scan was used for pregnancy diahnosis at around
day 60 after oestrus and to monitor fetal development there after at 2-week intervals.
From these 13 recipients, only one recipient was pregnant. And after 148
days pregnancy, Dolly was born safely with a birth weight of 6.6 kg.
Dolly's parentship
From Dolly's cloning procedure, it is clear that Superstar Dolly has no
father but fortunately that she has
3 "mothers" :
The nuclear donor adult cell - a Finn Dorset ewe.
The oocyte donor - a Scottish Blackface ewe.
The pregnancy recipient - another Scottish Blackface ewe.
Her nuclear genomic DNA came from the Finn Dorset ewe; her mitochondrial
DNA inherited from the oocyte donor Scottish Blackface ewe.
I would call the nuclear donor sheep as Dolly's "mother"
rather than her "delayed twin". Of course this is
not a classical genetic meaning of a mother.
"There will never be another you"!
So far, Dolly is the only mammalian cloned by an adult cell and she is
happy and healthy.
Dolly is unique, she has her own DNA set; she has her own character which
is formed by both gene-gene interactions and gene-environment interactions. And this
unique development of Dolly will continuing through out her whole life.
This stunning breakthrough certainly will have powerful implications in both basic and applied sciences.
However asexual reproduction in long terms should not replace the natural evolution process.
About the prospect of human cloning, Dr. Earl Watson's new book series
"The Era of Cloning Man?" will give the readers a detailed picture of the
possibility and the related scientific achievements.
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