July 23, 1998 NY Times

In Big Advance in Cloning, Biologists Create 50 Mice

By GINA KOLATA

Scientists from Hawaii said Wednesday that they have made dozens of adult mouse clones and even cloned some of those clones.

The announcement, coming after months of rumors, still stunned biologists when they heard the details. It means, they say, that advances in cloning are coming faster than even the most confident scientists had imagined.

The research team took just months to churn out clones of adult mice, following the announcement last year that the first clone of an adult animal, Dolly the sheep, was created in Scotland.

In Thursday's issue of Nature, Dr. Ryuzo Yanagimachi, 69, a biologist at the University of Hawaii, and his postdoctoral student, Dr. Teruhiko Wakayama, report on the first 22 mice that are clones, seven of which are clones of clones. They say they have made a total of more than 50 mouse clones.

The feat, scientists say, means that cloning an adult animal like Dolly is not a fluke as some have suggested. And with mice, they can study and perfect cloning in an easily available and familiar lab animal.

"Wow," said Dr. Barry Zirkin, who is the head of the division of reproductive biology at Johns Hopkins University in Baltimore. "This is going to be Dolly multiplied by 22."

In cloning mice, Yanagimachi and Wakayama defied conventional wisdom among biologists, who had long contended that mice would be perhaps the one mammal that was impossible to clone because of extraordinarily rapid development of the mouse embryo just after fertilization.

Dr. Lee Silver, a mouse geneticist and reproductive biologist at Princeton University, described the speed at which the cloning progressed as breathtaking. "It's absolutely incredible," he said. "He did all this in the past year."

The implications of the work were clear, Silver added. "Absolutely," he said, "we're going to have cloning of humans."

"If we follow scientific protocol," he added, "it could take 5 to 10 years before in vitro fertilization clinics add human cloning to their repertoires." The protocol would require that the method first be perfected and shown to be safe in mice and then monkeys. "But that does not mean that somebody would not do it without those steps," he said.

Zirkin said that human cloning "needs to be discussed," but he added that the main significance of Yanagimachi's announcement lies in the opportunities it creates for scientists to explore question about basic reproductive biology. "One of those questions, but only one," he said, "has to do with the cloning of humans."

The concept of genetically duplicating any person or animal by using the DNA of a single cell has captivated scientists and the public for decades.

But until last year, when the birth of Dolly was announced, most scientists had given up on the idea of cloning adults, although they generally agreed on the potential for cloning fetal cells. Even though every cell in the body has the same genetic material, cells of adults have reached their final state of development and never budge from it. Heart cells do not become liver cells, lung cells do not become brain cells.

To clone an adult, the genetic material from one of these cells would have to essentially go backwards in time and enter the state it was in when sperm first fertilized egg.

On Feb. 23, 1997, the world learned that Dr. Ian Wilmut of the Roslin Institute and Dr. Keith Campbell of PPL Therapeutics in Roslin, Scotland, had created Dolly by cloning from an udder cell of a six-year-old ewe.

Soon, critics emerged, led by Dr. Norton Zinder, a microbiologist at Rockefeller University in New York. Zinder questioned Wilmut's genetic evidence that Dolly was a clone. Moreover, he pointed out, it took 400 tries to produce Dolly and no other adult had been cloned. One success out of 400 tries is "an anecdote, not a result," Zinder said.

Wilmut responded to Zinder in two papers also published in Thursday's issue of Nature. Wilmut's group and an independent group that included the inventor of DNA fingerprinting, Dr. Alec Jeffries of the University of Leicester, compared DNA from the original piece of udder -- frozen at the Hannah Research Institute more than 100 hundred miles from the Roslin Institute -- to the DNA of the udder cells that were frozen in a test tube at the Roslin Institute and used to create Dolly to the DNA of Dolly. All the DNA sequences were identical, the researchers reported.

Dr. Davor Solter of the Max-Planck Institute for Immunobiology in Freiburg, Germany, wrote in an editorial accompanying the papers that the results "now lay these doubts" about Dolly "to rest."

But Zinder, in a telephone interview, said he still was not convinced. The investigators, he said, did not prove that the chunk of udder that they had stored at the Hannah Research Institute really was the source of the test tube of cells used to create Dolly. "They didn't keep proper records," Zinder said. "We don't know what that chunk of tissue was."

Zinder also questioned Yanagimachi's work. "I'm no mouse expert, but it looks to me like it's very very shaky," he said.

Those who are mouse experts did not share Zinder's doubts about the cloning.

"I am convinced," said Dr. John Eppig, an expert in mouse embryo development at the Jackson Laboratory in Bar Harbor, Maine. "Definitely. There is no question."

"The significance of this one is that it's incontrovertible," said Dr. Richard Schultz, an expert in early mouse embryo development at the University of Pennsylvania. It is, "a superb experiment," he said.

In his cloning experiment, begun just a year ago, Yanagimachi used one of Wilmut's key ideas, but varied his method.

Wilmut proposed that the secret to cloning was to put a cell into a resting state, so that it was not dividing, before using it to clone. He did this by starving the udder cells so they went into a state of hibernation. Then he slipped one of those cells into a sheep's egg whose own genetic material had been removed and gave the egg a shot of electricity to start the development.

In contrast, Yanagimachi and Wakayama started with three types of cells that were already in a resting state: cumulus cells, which cling to eggs like a thick smear of caviar; Sertoli cells, which are the male equivalent of cumulus cells, and brain cells. That experiment indicated that cumulus cells would be easiest to clone, so the scientists homed in on them and used them exclusively.

Yanagimachi and his colleagues injected the cumulus cell's genetic material into mouse eggs whose own DNA had been removed. They waited six hours to give the egg an chance to reprogram the cumulus cell's DNA and then chemically prodded the egg to start dividing. The process of reprogramming remains a mystery to the scientists.

All of the mouse clones were female.

Yanagimachi and his colleagues verified with genetic tests that their mice were indeed clones. Also, in one experiment, they used coffee colored mice for the cumulus cells, black mice for the eggs, and white albino mice as surrogate mothers. As predicted, the clones were coffee colored.

Despite the enthusiastic reception for the results of Yanagimachi's experiment among reproductive biologists, he had great difficulty publishing his paper. On Oct. 5, after cloning the first four mice, he submitted a paper to the journal Science, which rejected it without peer review, telling him it was "not of general interest," he said. Diane Dondershine, a spokeswoman for Science, said the journal's policy is not to comment on papers that were submitted.

Yanagimachi then he sent his paper to Nature, which forwarded it to two reviewers, one of whom asked repeatedly for additional proof. In March, the journal sent the paper to two more reviewers before finally accepting it last month, Yanagimachi said.

In the meantime, the scientists kept cloning.

Cloning has gotten so straightforward, Wakayama said in an interview, that he has now made more than 50 clones and could clone every day with no difficulty and turn out baby mice after the normal 20-day gestation period.

And lest anyone doubt that the clones exist, the scientists carried a cage of mice to New York to show them off, including a coffee colored adult mouse that was cloned, the clone created from that mouse, the black mouse that provided an egg for the cloning, and the white mouse that was the surrogate mother.

Already, a venture capitalist in Hawaii is setting up a consortium of companies and academic scientists to make the cloning of adult animals a commercial reality within a few years. But the investor, Laith Reynolds, the chief executive officer of Probio America, said, "We have no interest in cloning humans."

"Besides being the politically correct answer," he added, "we can't see any business in it."

Now, scientists say, the opportunity is here to figure out how cloning works, how to make it work better, and how to apply it.

For example, noted Dr. George Seidel, a cloning expert from Colorado State University in Fort Collins, scientists want to understand how an egg reverses the program of a cell's DNA. Once they figure that out, they might eventually be able to turn one cell into another.

"Let's say my pancreas was being destroyed for one reason or another," Seidel said. "If there are some cells in my body that can make a whole individual, they certainly can make a pancreas," he said. "It's pretty important, pretty potent stuff from a long-term standpoint," Seidel added.

And then there is the question of actually cloning people.

Human cloning, Seidel said on Wednesday, "is clearly more imminent."