New Experiment’s Success Confirms Cloning Works
Dolly the clone is no longer alone.
Resolving doubts about the authenticity of cloning, an international research team has produced three generations of cloned laboratory mice from adult cells, the group announced Wednesday. At the same time, two laboratories separately confirmed the unique genetic pedigree of the cloned sheep Dolly, whose creation triggered a worldwide furor.
Together, the new findings assure cloning a prominent role in the biology of the next century, experts said, but they do little to quiet international misgivings about the unprecedented control that the new technique promises over the ways that mammals--including human beings--reproduce themselves.
In research that may hasten the advent of commercial cloning, a group of Japanese, American and English researchers working at the University of Hawaii successfully cloned 50 animals from adult mouse cells, the scientists announced Wednesday. They used a new and perhaps more efficient cloning technique.
The team also demonstrated not only that their cloned mice can reproduce normally, like Dolly, but also that it is possible to create clones of clones by taking the new asexual reproduction technique of nuclear transfer through several generations without serious difficulty.
That finding--the first time clones have been produced from clones--means that “you can quite quickly make a colony of clones,” said embryologist Anthony Perry of the Babraham Institute in Cambridge, England, one member of the research team.
Their research marks the first time anyone has demonstrably duplicated the controversial cloning experiment that produced Dolly--by reproducing an animal from the nucleus of a mature cell taken from an adult.
Until now, except for Dolly, researchers have succeeded only in cloning cattle and other livestock from cells taken from embryos or fetuses, not from adults. In part because no one had been able to repeat the experiment, several prominent biologists had argued that the famous sheep was not a biological breakthrough but a laboratory error.
In research published today in the journal Nature, the Honolulu-based team led by Ryuzo Yanagimachi, an expert in reproductive biology at the University of Hawaii, put those doubts about cloning to rest.
“It worked very beautifully,” Yanagimachi said.
Robert J. Wall, an animal researcher with the U.S. Department of Agriculture, said, “The first and foremost aspect of this is that Dolly is not a fluke. Now the procedure of nuclear transfer has successfully produced offspring in another species using another kind of cell with a slightly different technique.”
“I don’t think there is going to be much in the way of questions whether Dolly is real,” Wall said.
“The importance of this report cannot be overemphasized,” said developmental biologist Davor Solter at the Max Planck Institute in Freiburg, Germany.
‘Exciting Results’
For the researchers most directly involved in cloning Dolly, the relief was audible.
Ian Wilmut, the embryologist in Scotland who led the team that created Dolly, has endured growing skepticism about whether his cloning work could be duplicated. He called the latest development “exciting results.”
Alan Colman, research director of PPL Therapeutics, the biotechnology firm based in Edinburgh, Scotland, that participated in the cloning of Dolly, called the latest research a “landmark.”
Harry Griffin, assistant director of the Roslin Institute, where Wilmut works, said, “We have never had any doubts that Dolly was derived from an adult cell.”
To create their families of cloned mice, the Hawaii group used a new technique pioneered by developmental biologist Teruhiko Wakayama from the University of Tokyo, the paper’s lead author. The cloned cells were combined mechanically, rather than being fused electrically, as Wilmut’s team did with the sheep cells that yielded Dolly.
Yanagimachi admitted somewhat sheepishly that his group only used the mechanical technique because the laboratory could not afford the expensive electro-fusion the Scottish researchers used to clone Dolly.
In addition to Wakayama and Yanagimachi, the Honolulu-based team also included Perry and Maurizo Zuccotti from the University of Pavia in Italy. Kenneth R. Johnson at the Jackson Laboratory in Bar Harbor, Maine, also worked on the experiments.
In all, the Hawaii team conducted a series of four cloning experiments--encompassing more than 1,240 attempts to create genetically identical mice from several different types of mature cells, including brain cells.
With a special micro-pipette, the researchers removed the nucleus of each adult mouse cell and quickly injected it into an egg whose own nucleus had been removed. They then gave the cloned egg cell five to six hours to adjust to its new state before chemically activating it so that the cloned cell would begin to divide and multiply like a normally fertilized embryo.
Once it did start to divide, the growing cluster of embryo-like cells was transferred from the lab dish to a surrogate female mouse, in the hope that it could be carried to term.
Although they had some limited success with all three cell types they tried to clone, they quickly discovered that only clones made from the raw material of cumulus cells, which surround the unfertilized eggs inside the ovaries of female mice, would result in a live birth.
Yanagimachi and Perry said they do not know why it is possible to clone some kinds of cells and not others. Cumulus cells are associated with reproductive tissue, but they have the same basic structure as any other cell and therefore there is no reason to suspect the cells might have special properties, the researchers said. Dolly was cloned from an udder cell.
The team’s first success--a cloned black mouse named Cumulina in honor of the cell type from which she sprung--was born on October 3 last year.
So there would be no question that the mice were clones, the researchers used strains that had different-colored fur. The mouse pups, either black or coffee-colored, matched the animals from which their cell nucleus was taken and not the mice that provided the donor eggs. The surrogate mothers were albino mice.
The researchers also subjected the clones to extensive genetic testing to make sure there was no mistaking their remarkable biological identity.
Of more than 800 embryos that actually began to develop in the surrogate female mice, only 17 were born, and seven of those died, the researchers reported. Even so, the technique appears to be more successful than the method used to produce Dolly, which produced only one lamb in 277 attempts.
Michael McClure, chief of the reproductive sciences branch of the National Institute of Child Health and Human Development, which partly funded the work, said, “This is a case where the right cell type, the right research design and the right technical expertise came together with great success.”
Legal Uncertainties
While it sets to rest any scientific uncertainty about the biological truth of cloning, the new finding may also trigger legal uncertainties over who has the right to license the potentially lucrative technique, which may find wide application in the genetic engineering of farm animals and in medical research.
The University of Hawaii has already licensed its cloning technique to ProBio America Inc., a subsidiary of an Australian biotechnology company. ProBio plans to apply it quickly in as many animal species as practical, company executives said.
The researchers also filed for a U.S. patent on their cloning technique in January, well in advance of any formal publication of their work.
It is unclear, however, whether the new technique is already encompassed by two patent applications filed last year by the Roslin Institute and PPL Therapeutics, raising the possibility that Dolly and Cumulina may find themselves facing off in a legal arena.
Earlier this year, the PPL firm gave an exclusive license for its own cloning technology to a new biotechnology company called Roslin Bio-Med, covering almost all biomedical applications.
On Wednesday, Colman in New York would not discuss the issue, but Griffin in Edinburgh said he was certain the Roslin Institute’s patent would prevail because it covered the basic cell characteristics essential to cloning, even if the actual injection techniques turned out to be different.
In the short run, several biotechnology executives said, cloning may provide more work for intellectual property lawyers than laboratory bioengineers.
In other research published in Nature on Wednesday, Dolly’s creators at the Roslin Institute and a group at the University of Leicester released new, more detailed genetic tests that confirm Dolly’s unique identity, in a further demonstration that the controversial white-faced sheep is indeed the product of a single cell taken from a 6-year-old adult ewe.
Several reproductive biologists had questioned publicly whether Dolly could have been mistakenly created from an embryo cell or from a fetal cell present in the bloodstream of the ewe, which apparently was pregnant at the time the original tissue samples were taken.
To buttress their claim to Dolly’s unique identity, the Roslin researchers took tissue and cells from the original ewe, which have been kept in cold storage at the Hannah Research Institute, and compared them through a series of genetic tests with a blood sample from the cloned sheep.
“All the best practices of forensic science have been applied to prove that Dolly was exactly what we said she was,” Colman said.
Hotz reported from Los Angeles and Goldman from New York.
To see a special report on the science of cloning and the issues surrounding its development, go to The Times’ Web site: https://www.latimes.com/cloning.
