SEND IN THE CLONES!

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 with sheep.

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.

WILMUT'S 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 the 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.

In 1987, however, Wilmut heard- in a now famous barroom conversation- that 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).

'My primary focus would probably be to. . . improve the method, whereas [basic scientists] would want to understand it.'

Although 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.

Wilmut 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- or whatever it was- with the change," he says.

Research 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- only 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.

Normally, 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.

They decided to use recipient eggs at three different positions in their cycles- but 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- the 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."

In March 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- passages 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 important.

But 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 is powerful."

The 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.

Wilmut 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- long thought to be a key to the regulation of transcription and thus development- affected 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."

Wilmut's 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'- I didn't have anything to say."

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