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Mouse cloning
is not forever

UH scientists fail to
make it work after
the sixth generation

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By Helen Altonn
Star-Bulletin

The historic cloning of mice by University of Hawaii scientists did not work after the sixth generation, the team reports in tomorrow's issue of Nature.

"The difficulty of yielding clones increased with each generation, and the experiment was brought to a halt when the single sixth-generation mouse was eaten by her foster mother," the researchers said.

The lead author was Teruhiko Wakayama, who did key work in 1998 on cloning of Cumulina, a female mouse, and in 1999 on cloning of Fibro, the first male mouse.

Wakayama formerly was an assistant professor at UH with "Team Yana," headed by Ryuzo Yanagimachi, professor of anatomy and reproductive biology in the John A. Burns School of Medicine. He joined Rockefeller University last year.

Yanagimachi said the findings reported in Nature represent work done two years ago that resulted in the pioneering "Honolulu Technique" of cloning generations of mice.

"We have old data so we keep publishing," he said. About 13 people were cited as authors on the paper, including six at UH.

The scientists cloned six generations of mice, taking genetic material in each case from somatic cells of the previous cloned generation.

The "Honolulu Technique" involves removing the nucleus from an adult cell and injecting it into an egg from which genetic information has been removed.

Wakayama and his colleagues said the cells of cloned mice show no signs of premature aging; some cells even showed signs of getting younger. By contrast, a study of the cloned sheep Dolly last year showed early wear and tear.

The UH scientists said the mice seemed mentally and physically normal until the sixth generation: "A fifth generation cloned mouse is alive and well in the rodent equivalent of middle age."

Yanagimachi said they did one series with four generations and another with six. It became more difficult with each generation, and they could not clone more than six, he said.

"We are studying why we can't continue. We are analyzing what went wrong."

The group found that protective structures, called telomeres, at the ends of the animals' chromosomes, usually reduced by cell division, appeared to lengthen slightly despite some predictions of telomere shortening.

"Any deleterious effects of cloning might be expected to be amplified in sequentially cloned mice," Wakayama's group said.

"Our results verify that telomere shortening is not a necessary outcome of the cloning process."

But since only 1 percent to 2 percent of reconstructed eggs yield live clones, the researchers said, "The possibility of selection for donor nuclei with the longest telomeres cannot be excluded."

Still puzzling, the scientists said, is whether genetic changes in the cells used to obtain nuclei may lead to adverse effects on the health of the clones.

"We cannot say cloned animals are entirely normal," Yanagimachi said.

But a collaborator from Japan is analyzing DNA from five generations of mice, he said. "We will get an answer in several months."

Yanagimachi has recruited two new faculty members for his team, now totaling 14, and he has three more positions to fill.

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University of Hawaii