In Big Advance in Cloning, Biologists Create 50 Mice

By GINA KOLATA
N.Y. Times
July 23, 1998

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."

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