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Nature 391, 825-826 (26 February 1998) | doi:10.1038/35934

Dolly researcher plans further experiments after challenges

Declan Butler

Dolly researcher plans further experiments after challenges

AP/DANIEL LUEDERT

Wilmut: "small possibility" that cloning was the result of inadvertently using a fetal cell.

Ian Wilmut, the Scottish researcher whose group last year reported the cloning of Dolly the lamb from the udder cell of an adult ewe, is planning further experiments to clarify the lamb's origin — and has offered to make frozen tissue from the donor ewe available to other researchers.

The move comes as many embryologists around the world have rallied to Wilmut's defence in rejecting challenges to Dolly's authenticity and playing down the significance of criticisms that his paper (Nature 385, 810; 1997) lacked unequivocal evidence that Dolly was derived from an adult cell.

The controversy has been triggered by a letter in Science describing Dolly as an "anecdote not a result", referring to the fact that it was the only successful birth out of several hundred attempts. The letter was written by Norton Zinder, a bacterial geneticist at Rockefeller University in New York, and Vittorio Sgaramella, a scientist at the University of Calabria in Italy (Science 279, 635; 1998).

The two researchers criticize what they claim was the poor characterization of the donor cells. They restate the possibility that Dolly could have originated from a stem cell — as noted in Wilmut's paper — and perhaps even from a circulating fetal cell, as the donor ewe was pregnant at the time the cells were obtained. They argue that the lamb's origins would have been better clarified by a genetic fingerprinting comparison with the donor ewe.

In fact, cells used in the cloning experiments were taken from a ewe that died three years before Dolly was born, and Wilmut's team did not directly compare Dolly's DNA with that of her clone. Ron James, managing director of PPL Therapeutics, which sponsored Wilmut's work, admits that in retrospect it would have been prudent to have taken more precautions.

But he explains that the mammary gland cells used had not been better characterized because they were prepared for other studies aimed at increasing expression of milk proteins, and had not been created with nuclear transfer experiments in mind.

This explanation cuts little ice with critics such as Richard Gardner, an embryologist at the University of Oxford. "I don't believe this was an 'after-tea' experiment," he says. "It should have been better documented." Gardner describes the absence of a direct comparison between Dolly and her clone as a "staggering omission", leaving open the possibility of a mix-up in the cells used. Others who have criticized the relative lack of evidence include the Nobel laureate Walter Gilbert of Harvard University.

Wilmut has argued that a mix-up of cell cultures was unlikely, given that Dolly is a Finn Dorset ewe, and that no other Finn Dorset cells were being cultured in the laboratory. But he admitted last week that, although the prospect is highly unlikely, there is a small possibility that Dolly could have originated from a fetal cell, and that he may repeat the experiments.

In response to the criticisms, PPL has asked an unnamed US university to verify that Dolly's mitochondrial DNA came from the Scottish Blackface breed of sheep used as a source of unfertilized eggs, and an unnamed British team to compare Dolly's DNA with that of stored samples of the udder cells.

A definitive answer should come from a comparison of Dolly's DNA with tissues from the donor ewe. Wilmut says frozen samples were kept, and are being studied by an independent group. "Other samples will also be available to interested parties if they wish," he says.

Many cloning scientists have come to Wilmut's defence. James Robl, a scientist at Texas Transgenic Nuclear Transfer Calves, describes Zinder and Sgaramella's criticisms as "nitpicking". He says they are "out of touch" with cloning research. Robl argues that, far from being an "anecdote", one birth from 277 attempts is a "very respectable result in this field where there are many steps, each of which take their toll on efficiency".

Robl dismisses as highly improbable the possibility that Dolly might have originated from a fetal cell, given that the sheep placenta contains many layers of tissues separating the fetus from the mother's blood.

Ken White, a cloning scientist at Utah State University, says: "Retrospectively you can say that we can't go back and check, but it is always easy to look back. I don't think that Ian Wilmut needed to do more." He adds: "I wouldn't be so quick to run up red flags saying that no-one has been able to reproduce the work. It's too soon. If by 2000 no-one has duplicated it, I'll start being concerned. But am I concerned in 1998? Not at all."

Zinder and Sgaramella argued that their scepticism had been provoked by the fact that no-one has reproduced the results. But David Wells, a scientist at the Ruakara agriculture research centre in New Zealand, who has reproduced Wilmut's work on cloning from fetal fibroblasts, and is engaged in cloning adult bovine cells, describes the controversy as "premature".

Wells says it is unfair to expect rapid confirmation of the results, given the time and money needed for such experiments, the long gestation periods of farm animals and the seasonality of their reproduction.

Bob Moore, who has recently retired as deputy director of the Babraham Agricultural Institute near Cambridge, says he is not surprised that other scientists have not yet repeated Wilmut's finding, given the difficulties of obtaining success. "I think people are having difficulties reproducing the results, but you only have to look at [the low efficiency of] Wilmut results to predict that they should be having difficulties."

Moore describes the controversy as a "storm in a teacup". He points out that Dolly is part of a growing body of science that has witnessed a logical progression from the production of clones from embryos, and differentiated fetal fibroblasts, to the routine generation of embryos from adult somatic cells, even though, apart from Dolly, none have gone to term.

Jean-Paul Renard, a scientist at the French national agricultural agency INRA, who is attempting to clone cattle from adult cells, is even more confident. "Our work leads us to think that Dolly is a reality, and will be reproduced," he says.

James argues that, contrary to popular belief, few groups are racing to reproduce the Dolly experiment. For commercial purposes, cloning from fetal fibroblasts is more efficient, while their genetic modification is relatively straightforward, he says. "It delivers what we need — a genetically modified animal." Wilmut's group has so far not attempted to repeat the experiment simply because "Dolly is not commercially useful," James argues.

"You are talking at least $1 million" to repeat the Dolly experiment, says Robl. He is also pursuing cloning from fetal fibroblasts as the most immediately promising commercial option, while carrying out research to develop more efficient systems for cloning from adult cells.

Zinder remains unconvinced by such arguments, asserting that the extensive media and political reaction to cloning has obscured the fact that "the emperor has no clothes". He says Wilmut should have been required to repeat the result before his paper was published.