On March 13, Scottish researcher Ian Wilmut
filled Masur Auditorium and several overflow rooms with scientists
and others eager to learn of his coup defeating the dogmas of mammalian
cell differentiation. On the same day, the National Bioethics Advisory
Commission assembled for its first meeting in response to a presidential
directive that it produce a report and recommendations on the state
of the art and the legal and ethical ramifications of human cloning
research - a directive inspired by Wilmut's cloning achievement
The NIH Catalyst seized the moment, dispatching
NIDCD Fellow David Ehrenstein to conduct a one-on-one interview
with Wilmut after he'd addressed the NIH masses, Scientific Editor
Celia Hooper to snap photos of Wilmut engaged in this dialogue,
and Managing Editor Fran Pollner to cover the ethics panel deliberations,
the results of which may well shape the types of cloning-related
research NIH scientists will be allowed to pursue. Our reports-
and a technical review by NICHD's Alan Wolffe, whose own research
is connected to the issue at hand- follow.
LUCKY LAMB SHEPHERDS
IN NEW ERA
OF DEVELOPMENTAL RESEARCH
by David Ehrenstein
Ian Wilmut: A Man On the Go
Ian Wilmut admits to some
good fortune when he created the lamb named Dolly, the first mammal
to be cloned from the DNA of an adult animal. He paraphrases the
British scientist Peter Medawar on the definition of a good experiment:
"You have to go far enough forward that it really does add
to knowledge, but not attempt to go so far forward that it doesn't
work, or you don't understand it. . . . I think we got lucky with
this one, and it's gone a long way," he said in a recent interview
with The NIH Catalyst.
But Wilmut was not trying to make history.
Since the mid-1980s, his goal had been to manipulate the genes of
farm animals as easily as other scientists modify mouse genes. With
such techniques, drug companies could genetically manipulate livestock
to generate large quantities of human drugs or hormones in the animals'
milk, or perhaps create a scrapie-free strain of sheep. So it was
actually attempts to manipulate the sheep genome, not clone it,
that led Wilmut and his colleagues to produce Dolly.
A decade ago, Wilmut's lab and others had tried to culture the
livestock equivalent of mouse embryonic stem (ES) cells-
undifferentiated cells that are
traditional starting point for transgenic work in lab animals. But
the sheep cells wouldn't remain ES-like for more than three cell-division
cycles, or "passages"; after that, their morphology changed,
showing signs of differentiation. The traditional transgenic techniques
require cells that remain undifferentiated much longer.
however, Wilmut heard- in
a now famous barroom conversation-
Steen Willadsen (now of the St. Barnabas Medical Center in West
Orange, N.J.) had managed to produce calves with a new procedure:
Willadsen took cells from the interior of many-celled embryos (blastocysts)
and transferred their nuclei into eggs whose own nuclei had been
removed (enucleated eggs).
Willadsen had previously accomplished nuclear transfer from 16-cell
embryos, it was generally believed that nuclei from older embryos,
which have begun transcription and differentiation, could not be completely
"deprogrammed" by the egg cytoplasm to restart normal development.
primary focus would probably be to. . . improve the method,
whereas [basic scientists] would want to understand it.'
realized that the sheep cell lines he had already made, which were
also derived from interior blastocyst cells, might be useful for
manipulating genes after all, if he used them as nuclear donors.
He hoped to select donor cells that had been through several rounds
of cell division, allowing enough time "to be able to do gene
targeting to select the cells with the [desired] change . . . and
then do nuclear transfer and therefore get your calf or lamb-
whatever it was- with
the change," he says.
in large animals takes time; the gestation of a sheep is five months.
And after two sheep seasons of testing, the longevity of the ES-like
cells still appeared to be a limiting factor-
early-generation donor cells, through passage 3, produced any lambs.
But Wilmut and his colleagues were persistent. They decided to manipulate
the cell cycles of the donor and recipient cells more carefully,
and that turned out to be critical.
the cell cycle consists of four phases: M (mitosis), G1,
S (DNA replication phase), and G2,
although early embryonic cells and stem cells skip the "gap"
phases, G1 and G2.
If a cell is deprived of serum (which includes growth factors),
it exits the cycle at G1 and
slips into a quiescent state, known as G0,
where it can remain for months or years, until growth factors are
added back to the medium. It was known that successful nuclear transfer
required some coordination of the cell cycles of donor and recipient,
and the later-passage cells allowed Wilmut's group to manipulate
the more complex cycles of differentiated cells.
decided to use recipient eggs at three different positions in their
to use donor cells only in G0.
In this case, convenience dictated the choice: cells could be held
in G0 as long as necessary, whereas
G1, another possibility, would
require precise timing. It was during these experiments that Keith
Campbell, the cell biologist in the group, pointed out that G0
might be advantageous for another reason-
lack of transcriptional activity in the cells might allow such a
nucleus to be reprogrammed more easily in the egg. At that point,
the scientists realized the possible significance of G0
and stopped discussing their work with others. "In our country,
if you've gone public with information, you can't patent it. . .
. If we had talked to anybody about that, we would've lost the patent."
of 1996, Wilmut's group published a paper in Nature showing
their ground-breaking results: five lambs had been born, the first
mammals ever generated from cells of an established cell line-
6 through 13. This achievement suggested that gene manipulation
of farm animals would indeed be possible. Putting the donor cells
into G0 appeared to be the critical
new trick; the phase of the recipient egg turned out to be less
not everyone was convinced. Some researchers credited the nuclear
transfer and cell-cycle manipulation, while others thought he had
simply used the right cells, Wilmut recalled. "And so what
we were trying to do this year [in 1996] was to take another embryo
population, a fetal population, and an adult population [of donor
nuclei] and just see how powerful the nuclear transfer technique
was. And the answer is yes, . . . it's the nuclear transfer that
result from the adult cell line was, of course, the most famous
of the three, but Dolly was almost an afterthought. "We were
having a lot of success with the new [donor] embryo cells and .
. . fetal cells; then we thought, well, let's stick the adults on,
as it were. But the original
intention 18 months ago [was] to just do embryo and fetal cells."
In fact, the adult mammary epithelium cells they used were in the
lab's cold room only because another group was studying the manipulation
of milk protein genes.
is now eager to improve the efficiency of nuclear transfer, which
yielded only one lamb out of 277 adult nuclei that were transplanted.
One of his first steps will be to try adult cell types
other than mammary epithelium. There are also many basic science
questions remaining: How is the chromatin structure-
thought to be a key to the regulation of transcription and thus
by the nuclear transfer? What specific proteins in the egg are involved
in "reprogramming" nuclei derived from mature animal cells?
What's special about the G0 chromatin?
But Wilmut probably won't investigate such basic questions in his
lab. "I guess my primary focus would probably be to try and
improve the method, whereas [basic scientists] would want to understand
it, I suspect."
work has likely opened up a whole new realm of investigation for
both basic and applied researchers. That can be exhilarating, but
it can also be frustrating. So many unknowns surround these experiments,
Wilmut acknowledged, that he often finds himself unable to answer
questions. "I went into a lab meeting [at NIH] . . . and I
almost wrote on the board 'we don't know'-
didn't have anything to say."