DOLLY is one of a kind. That may be her claim to fame, but it is also the
biggest problem now facing her creators. While the majority of cloning experts
believe in Dolly, many prominent biologists, including some who sat on President
Clinton’s cloning advisory committee, argue that there’s just not enough proof
that she’s for real. And their scepticism is fuelled, they say, as each known
attempt to repeat the Dolly experiment fails.
To make a tense situation worse, cloning research has all but gone
underground as investigators try to dodge accusations of poor-quality research,
protect commercially valuable findings, and avoid the prying eyes of the
media.
But that doesn’t mean that Ian Wilmut at the Roslin Institute, Keith Campbell
of PPL Therapeutics in Edinburgh, and other team members have been taking the
criticisms lying down. Indeed, they say that they now have fresh results to
support their claim that Dolly is an adult clone, and they are promising to
release the evidence that will silence the critics any day now.
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“Our [latest] tests have confirmed that Dolly is the real thing. We are
absolutely confident that any tests by any other group will confirm that Dolly
is the first clone of an adult animal,” Wilmut told New Scientist at a
meeting in Melbourne, Australia, last month.
Some scepticism is to be expected, considering biology’s recent history.
Early this century, biologists watched what happened when they moved cells and
cell nuclei from one part of an embryo to another at different stages of
development. Those findings laid down the law: once cells take on specialised
tasks, their fates are sealed and they can’t then become other types of cells,
including embryo cells.
Impossible dream
A series of experiments on frogs in the 1960s and 1970s overturned that
dogma, but, ironically, also convinced many biologists that cloning adult
animals was an impossible dream. John Gurdon of the University of Cambridge
injected nuclei from the intestinal cells of tadpoles into eggs whose own
nuclear material had been destroyed, producing healthy, fertile frogs. But when
Gurdon used cells from the foot of an adult frog as his nuclear donor, only a
very few of the embryos grew into healthy-looking tadpoles, and not one made it
to froghood.
For PPL’s Alan Colman, who worked in Gurdon’s lab at the time, these results
were a sure sign that adult animals could never be cloned. “I saw how hard they
tried to get adult frogs and failed. It coloured my views,” he says. It took
nothing less than Dolly to change his mind.
But a few were inspired by Gurdon to move the science of cloning forward. In
the 1980s, researchers managed to clone sheep and cows from cells of 8 to
16-cell embryos which had not yet become specialised. And just the year before
Dolly’s debut, the Roslin team made a splash when they cloned Morag and Megan
from embryonic sheep cells that had grown for several generations in a lab dish,
specialising into epithelium, a class of cell that includes skin.
For their next step, the team wanted to push the technique a little further
by cloning from even more specialised tissue, cells from 26-day fetuses. If they
succeeded they would have a virtually unlimited number of cells to use for
genetic engineering, which was the goal of their project
(see “We ask, they answer”, p 26).
Almost as an afterthought, they decided to go for the full
monty, with completely specialised udder cells from a six-year-old ewe obtained
from the Hannah Institute in Ayr. With little expectation of success, they
didn’t worry that the donor ewe was dead, and they made no special effort to set
aside extra samples of her tissue for later genetic analysis. They would later
be criticised for these decisions. In retrospect, says Colman, the team would
have been wise to “deposit some of the donor tissue with a respected third
貹ٲ”.
At the time, the most important thing about the frozen udder cells was that
the researchers could thaw them, grow them in a flask, and control their supply
of nutrients. That allowed them to put the cells on a near-starvation
diet—a trick that Campbell believes was key to their previous cloning
successes.
As cells start running out of nutrients, they slip into the genetic
equivalent of suspended animation, a state called G0 where they no longer copy
DNA or divide, and turn off all but their most vital genes. The idea is that the
cells develop amnesia, forgetting their past commitment to be a particular type
of tissue. When the cell’s nucleus is transplanted into an egg whose nuclear
material has been removed, and is awakened by the egg’s reprogramming machinery,
it is supposedly more likely to take new instructions.
Brainwashing
Campbell now admits that it is a mystery precisely how this molecular
brainwashing takes place, and which are the crucial steps for rejuvenating
specialised cell nuclei. “This technology is in its infancy and we know we’re
just scraping the surface of our understanding,” he says. Nonetheless, the trick
seemed to have worked at least once. Dolly was born.
Other researchers quickly followed their lead. Although their work has yet to
appear in the scientific literature, cloning specialists at biotech companies in
the US and working for the French government have since cloned cows, such as
Gene, and the now-deceased Marguerite, from specialised fetal cells.
Clones from adult cells, however, have been more elusive. In August 1997,
Infigen, the Wisconsin biotech company that produced Gene, proudly announced
that its cows were in the late stages of pregnancy with adult clones. None of
these bovine Dollys has yet materialised, even though a cow’s gestation takes
only nine months. “That work is still moving along,” is all Michael Bishop,
Infigen’s vice-president of research, will say, claiming he is restricted from
giving more information by commercial agreements with ABS Global, Infigen’s
parent company.
“I have no doubt adult cloning can be done,” says James Robl of the
University of Massachusetts at Amherst. According to Robl, his group has found
that clones from adult cells tend to die in the womb at around 50 days, about a
week before organ formation is complete. He thinks he knows why it’s happening,
but refuses to give details for fear of tipping off his competitors.
Still, even as the flurry of excitement over Dolly’s birth announcement in
February 1997 was dying down, doubts about her significance started to bubble to
the surface. One note of dissent was voiced only three months later in the
report on cloning ordered by President Bill Clinton from the National Bioethics
Advisory Commission (NBAC), a committee of doctors, ethicists, lawyers,
scientists and theologians.
Before launching into lengthy discussions about the religious, ethical, legal
and policy implications of adult cloning, the report summed up feelings about
Dolly with a remarkably unenthusiastic comment: little was learnt about cloning
of specialised cells, the report said, because “Dolly could have been derived
from a less-differentiated cell in the [udder culture] population”. And the
report’s authors noted that, unlike Gurdon before them, Dolly’s creators didn’t
perform tests to show that adult genes were active in the cells they chose to
clone.
Privately, the criticisms went further. One of those authors, Carol Greider
of Johns Hopkins University School of Medicine in Baltimore, says that many
scientists were surprised that Wilmut’s group had published the Dolly experiment
before showing it could be reproduced.
The NBAC’s quiet objections found a very public voice this January, when
Norton Zinder of Rockefeller University in New York and Vittorio Sgaramella at
the University of Calabria in Italy blasted the Wilmut team in a letter to
Science (vol 279, p 635). Their list of criticisms is now well known to
every reproductive biologist.
Foremost is the charge that the genetic analysis Wilmut and his colleagues
used to support their claim was inadequate. One problem is that they only
compared Dolly’s genes with the genes of the cells in culture, not directly with
the ewe from which these cells were derived. That proves that Dolly is related
to the culture, but not that all the cultured cells come from an adult sheep
rather than from undifferentiated cells. Wilmut’s group even admits in its
original paper that its culture dishes contained a selection of other,
non-mammary cells. And tubes do get mixed up, a scenario that at least seems
possible in light of the difficulty in repeating the Dolly experiment.
Relative values
A second problem is that the Wilmut team used only four regions of DNA to
compare Dolly with the cultured sheep cells. More seriously, they don’t know how
likely it is that two unrelated sheep will share those same regions. Without
that knowledge, the critics argue, it’s simply impossible to say how related one
animal is to another or to a cultured cell. At worst, it could be like claiming
two people are twins because they both have A-negative blood—you could
only know that argument was flawed by knowing how often A-negative blood pops up
in the population at large.
Zinder and Sgaramella’s next grievance was that Dolly’s “mother” was pregnant
when cells were taken from her udder. In some animals, fetal cells enter the
mother’s blood and could possibly get into the udder. In other words, a wayward
fetal cell could be Dolly’s true parent.
A few weeks ago, Wilmut announced that new work has undermined the theory of
a rogue fetal cell floating in the blood of sheep. “Our collaborators,” he told
a public meeting on 18 February at the Smithsonian Institution in Washington DC,
looked for fetal cells in the blood of pregnant ewes. “There is no evidence of
that happening in sheep.”
Adding tension to the debate came the rumour that, more like comic book
absent-minded professors than serious scientists, the Wilmut team had lost track
of the remaining udder cells from Dolly’s “mother”—a rumour Wilmut and his
colleagues vigorously deny. And to prove their point, researchers at PPL have
acquired more of the original udder tissue from the Hannah and analysed it. The
four regions of DNA are identical to Dolly’s and to the udder cell culture, says
Colman. That still fails to take into account how often identical versions of
the four regions occur in all sheep, but at least the result is in the right
direction, shoring up the idea that Dolly is an adult clone.
One in a million
The team has also sent some fresh udder tissue to independent researchers in
the US—Wilmut and his team decline to specify who—to do more
extensive genetic analysis. “Then we’ll be able to say the chance of a random
match is less than one in a million or ten million,” promises Colman. One lab
will compare Dolly’s genes to the genes in the udder tissue. Another lab will
check out her mitochondrial genes. Because the egg is the major contributor of
mitochondria to the embryo, at least some of Dolly’s mitochondria should be
similar to the Scottish blackface egg donor.
And just to hedge its bets, the Wilmut team has asked another unnamed lab to
look at the length of Dolly’s telomeres—the DNA at the end of chromosomes
that tends to shrink as an animal ages. Dolly may turn out to have shorter
telomeres than your average sheep, because her “mother” cell incurred six extra
years of telomere loss. That would be excellent evidence that Dolly is the
offspring of an adult cell. If, on the other hand, the telomeres are a regular
length the result will neither prove nor disprove that she’s an adult clone, as
eggs are rich in the enzymes that patch up telomeres.
While the tussle over Dolly’s origins may be warranted, that doesn’t mean the
reputation of cloning science or Wilmut’s team rests on her alone, says
developmental biologist John Eppig of the Jackson Laboratory in Bar Harbor,
Maine. “Cloning is progressing and what Ian Wilmut’s group achieved before Dolly
was already a remarkable scientific accomplishment.”
For their part, the Scottish scientists seem confident that the new genetic
analysis will vindicate all their work. “It will prove unequivocally that
Dolly’s genetic parent was an adult cell,” says Colman.
But even that extra effort won’t completely satisfy the fiercest critics.
“Even if Dolly is real, there are so many important scientific issues that
weren’t, and now can’t, be addressed,” says Zinder. How, for example, do her
physiology, immunology, or even appearance, compare with her long dead
twin-cum-mother’s, he asks. “You want to see them side by side.” And there is
only one way to do that: repeat the experiment with the wisdom of hindsight.
What Dolly really needs to secure her place in history is some company.
Clone watching
Who will be first to create the next Dolly?
Our top tips:
-
Jean-Paul Renard at France’s national agricultural research institute
(INRA) in Jouy-en-Josas, near Paris, has a cow halfway through gestation,
pregnant with a clone of a two-week-old calf. The expectant mother is reportedly
doing well. -
James Robl of the University of Massachusetts in Amherst is the most open
about the technical difficulties of cloning adult animals. He’s banking on a
systematic approach to the problems as the way to get results. -
Alan Trounson of the Institute of Reproduction and Development at Monash
University in Melbourne, Australia, helped pioneer in vitro fertilisation in
sheep, and then in humans. Now, he’s eager to make cloning livestock a practical
proposition. -
For many months, Ian Wilmut insisted he had no plans to create another
Dolly, but recently he told New Scientist: “We and lots of other labs
will work with a variety of different adult cells. We would expect to see many
new results over the next year or so.” -
Infigen’s Michael Bishop steadfastly claims that his company will “very
soon” be celebrating the birth of a clone of an adult cow. But things can’t have
been going smoothly: Infigen has been making that claim for at least nine
months, the gestation period of a cow.