Dolly was lucky: scientists warn that cloning is too dangerous for people.

As scientists consider whether human cloning can be safe, the stories of two sheep, one famous and one dead, illustrate the dream and the danger.

One tale centers on the photogenic Dolly, the first animal ever cloned from an adult mammalian cell (SN: 3/1/97, p. 132). While investigators continue to study the 5-year-old sheep for late-developing abnormalities, such as premature aging, Dolly has given birth to normal lambs and is by all accounts healthy. She may be a bit overweight, but that's because reporters have fed her so much, jokes Alan Colman of PPL Therapeutics in Edinburgh, Scotland, which funded the creation of Dolly.

Less well known and lacking a cute public name was a cloned ewe born not too long after Dolly. It had no obvious physical abnormalities at birth and was an active lamb, but it panted all the time, recalls lan Wilmut of the Roslin Institute in Edinburgh, whose group cloned both Dolly and the second ewe. The respiratory problem was so severe that researchers within a few weeks decided to euthanize the hyperventilating animal. An autopsy showed that its lungs had not developed properly.

Wilmut says that this second ewe's fate ought to make those who would clone people think again. "Who would be responsible for a child born with an abnormality like that?" he asks.

Although Dolly's birth has inspired a few maverick researchers to want to make human cloning a reality, the often-disastrous results of animal cloning have convinced many scientists that an effort to clone a person is unthinkable at this time.

"Based on the plausible outcomes, it's ridiculous to move forward with human cloning," says Don Wolf of the Oregon Regional Primate Research Center in Beaverton, who is working to clone monkeys (see box, page 251). "It's totally irresponsible."

In August, a scientific and frequently emotional discussion on the feasibility of human cloning played out in public at the National Academy of Sciences (NAS) in Washington, D.C. (SN: 8/18/01, p. 105). There, before a panel considering whether to recommend a ban on human cloning, more than a dozen scientists described their successes and failures at cloning mice, sheep, goats, and cows. As they discussed possible explanations for what goes wrong, the scientists often focused on evidence of abnormal gene activity during clones' development. And almost all concluded that cloning a person would be unsafe.

But not everyone did. Three proponents of human cloning defended their plans and vowed to continue. They argued that scientists have more knowledge about reproduction in people than in most other species, and people may not be susceptible to some of the problems that have arisen in cloned animals.

"We need to proceed with human cloning," says Brigitte Boisselier, a chemist and director of a cloning company formerly called Clonaid, whose location she refuses to reveal. "I believe it's a fundamental right to reproduce the way you want."

At the meeting and in scientific publications, researchers have documented a litany of problems that plague animal cloning. Many of the cloned embryos develop so abnormally that they don't even make it out of the petri dish alive.

Despite its failure rate, the cloning process is straightforward (SN: 4/5/97, p. 214). First, investigators obtain an egg cell from an animal and remove its nucleus, the sac containing almost all the egg's DNA. They replace that nucleus with one from a cell of the animal they wish to copy. Usually, this is done by fusing the nucleus-lacking egg with the donor cell.

Finally, a jolt of electricity or some other stimulus tricks the egg into dividing as if it had been fertilized by a sperm. Once the growing embryo has reached a multicell stage known as a blastocyst, it's ready to be transferred into the uterus of a surrogate mother.

Jonathan Hill of Cornell University, who has cloned cattle, notes that about one-third of the cloned embryos that are implanted don't survive even the first month of a cow's normal 9-month gestation period. Of those that do, another half die in the next month or two, apparently because of abnormal placental development. This prenatal die-off continues through birth.

"The placenta is not supplying nutrients, and the fetus starves," says Hill.

The same pattern of spontaneous abortions holds true for cloning in other species. "The losses are extraordinarily large and happen at all stages of gestation," says Colman.

Making it to birth is no guarantee that a clone will survive. Hill notes that new-born cloned calves frequently emerge in bad shape. Some have skeletal abnormalities. Many suffer a variety of lung and heart problems.

Hill estimates that 25 to 50 percent of clones are oxygen-deprived at birth. Some can be saved, but many die.

Cloned cows, sheep, goats, and mice also often display what scientists call large-offspring syndrome. Internal organs, limbs, and overall body are oversized, and the newborns are sickly. The large fetuses can also place a mother at risk during delivery.

Colman, however, says that the sorry state of cloned animals has been exaggerated. At the August meeting, Colman presented data from several research groups--including his own--showing that in some cases, 95 to 100 percent of cloned pigs, cows, and sheep that made it to birth were thriving. "Many cloned animals are healthy," he says. "But because most of us here abhor the idea of human reproductive cloning, there's an extrapolation of results to indicate that [we] never get any healthy clones."

Colman also offered the scientists a sense of cloning's efficiency by comparing it with human in vitro fertilization (IVF). He noted that many human embryos created through IVF, like cloned embryos, don't make it to the blastocyst stage.

In one set of published data, only 8 to 12 percent of the human embryos created with IVF resulted in a live birth, he says. Some groups that are cloning cattle have achieved comparable efficiencies, Colman noted.

So, what exactly goes wrong when cloning fails? Because Dolly and some other cloned animals have begotten normal offspring, scientists don't think that cloning introduces permanent mutations into an animal's genes.

Consequently, biologists have begun to focus on the regulation of gene activity in cloned embryos. When a nucleus from an adult cell, say a skin cell, is placed inside an egg cell, its DNA must undergo dramatic changes before it's ready to create a new animal. Skin-specific genes must turn off, for example, and genes that drive embryonic development must begin to turn on, each one at exactly the right time.

Scientists refer to this transformation as the reprogramming of the nucleus. Incomplete reprogramming is the main reason that cloned embryos fail so often, they suggest.

Several recent studies have suggested a problem with methylation, a chemical modification that usually shuts down gene activity. As cells begin to specialize into adult tissues, methylation seems to inactivate genes that are no longer needed. For the DNA in an adult nucleus to guide the development of a clone, its existing methylation pattern must return to an embryonic state.

In the June NATURE GENETICS, however, South Korean scientists reported that methylation patterns in cloned bovine embryos are frequently quite different from those observed in normal embryos.

A few months earlier, a research group that included some of Dolly's creators focused attention on a gene encoding a protein called insulin growth factor 2 receptor (IGF2R). The gene normally shows a trait known as maternal imprinting--only the copy inherited from the mother is active in an offspring. There are also paternally imprinted genes, and scientists believe that methylation plays a large role in maintaining the imprinted status of a gene. The parental gene that is methylated lies dormant, leaving the other parent's copy of the gene active in the offspring.

The Roslin Institute's Lorraine Young and her colleagues examined the activity of the IGF2R gene in sheep fetuses created through IVF, which like cloning often results in large-offspring syndrome in sheep. Compared with normal-size fetuses, those showing signs of the syndrome had 30 to 60 percent less activity of the gene the team reported in the February NATURE GENETICS. Moreover, DNA regulating the gene's activity showed less methylation than normal.

Since IGF2R normally plays a growth-suppressing role within the fetus, the scientists concluded that reduced activity of its gene could be responsible for large-offspring syndrome both in IVF and cloned embryos.

There are about 40 known imprinted genes in a person. In general, genes in which only the paternal copy is active promote fetal growth, while genes in which just the maternal copy is active limit it (SN: 5/15/99, p. 312).

The profound influence of imprinted genes on fetal growth has made them prime suspects for many of the developmental abnormalities that afflict cloned animals. Some scientists have even looked at these genes in cloned animals that show no obvious defects.

Rudolf Jaenisch of the Whitehead Institute for Biomedical Research in Cambridge, Mass., and his colleagues recently examined seemingly normal adult mice that had been cloned by placing the nucleus from an embryonic stem cell into a mouse egg. The researchers observed inconsistent methylation and activity among the handful of imprinted genes that they tested, not including that for IGF2R, Jaenisch reported at the NAS meeting and in the July 6 SCIENCE.

"Even apparently normal clones have an abnormal regulation of many genes," he contends. "Completely normal clones may be the exception."

Other scientists questioned this conclusion. They note that cloning typically uses a nucleus from an adult cell, not from an embryonic stem cell. Therefore, the genetic chaos Jaenisch observed may not be pertinent to the more common methods of cloning.

Jaenisch "has laid down the gauntlet for us to prove this," says Colman.

Even as investigators probe the reasons that animal cloning fails so often, another question has arisen: How relevant is that research to human cloning? After all, although animals created through in vitro fertilization sometimes suffer large offspring syndrome, there's no evidence that human test-tube babies do.

Randy L. Jirtle of Duke University Medical Center in Durham, N.C., and his colleagues have now added a new element to the debate. In the Aug. 15 HUMAN MOLECULAR GENETICS, the investigators report that the gene for IGF2R is not imprinted in primates, even though it is in rodents, pigs, sheep, and other animals that researchers have cloned. So, people have two active copies of this growth-suppressing gene instead of just one.

Jirtle's team is focusing on the finding's implications for cancer, but it might explain why people are less susceptible than some animals to fetal overgrowth. "This marked species difference in IGF2R gene imprinting indicates that humans may be easier to clone than nonprimates," Jirtle says, who adds that he's not advocating such attempts.

Yet abnormal activity of the IGFR2 genes may not be the only factor contributing to large-offspring syndrome. Indeed, Jaenisch's group has observed that some oversize mouse clones have normally imprinted IGFR2 genes.

In addition to Boisselier's company, which is affiliated with the UFO cult known as the Raelians, two individuals have announced that they plan to collaborate on an effort to clone people. Severino Antinori of the International Associated Research Institute in Rome has a long history of work in assisted reproductive technology. He recently received both recognition and condemnation for help-ing postmenopausal women, such as a 63-year-old grandmother, become pregnant with the use of donated eggs. Antinori intends to work with reproductive physiologist Panayiotis Michael Zavos of the Adrology Institute in Lexington, Ky.

Zavos argued at the August meeting that the animal data present an overly pessimistic view of cloning for several reasons. The animals cloned often come from inbred strains, which he speculates make cloning more difficult. Furthermore, the cloned animal embryos chosen for implantation, Zavos says, do not undergo any kind of screening process to weed out the ones unlikely to succeed.

Antinori adds that animal cloners may not be using the optimum culture conditions for initially growing the embryos.

By applying decades of expertise in IVF, it's possible to make cloning as safe and efficient for people as are other reproductive technologies, Antinori and Zavos contend. They point out that some IVF physicians already screen human embryos by plucking out one cell and checking it for specific mutations before placing the rest of the embryo in a woman's uterus.

Furthermore, in vague comments that Boisselier declined to explain more fully, she suggested to the NAS panel that her research group had developed ways to guarantee cloning safety by checking the on-off status of imprinted genes in a human embryo.

Reproductive biologist Alan Trounson of the Monash Institute in Clayton, Australia immediately derided Boisselier's claim as "ludicrous."

First, he says, imprinted genes are not the only genes misregulated in clones. Second, he and most of the scientists at the NAS meeting agreed, today's technology isn't advanced enough to check dozens or hundreds of genes at one time. When physicians now do preimplantation diagnosis, it typically focuses on identifying mutations in a single gene. Finally, many of the genes that may cause a problem don't become active until after the implantation of an embryo.

"At present, there is no way to predict whether a given clone will develop into a normal or abnormal individual," Jaenisch concludes.

The desire to guarantee that a human clone be healthy may reflect a philosophical difference between people favoring and opposing attempts at human cloning. Pointing to the high rates of spontaneous abortions and birth defects that plague natural pregnancies, Zavos argues that human reproduction is intrinsically risky.

Of course, more than safety arguments enter discussions of human cloning. Some people object to cloning out of religious, ethical, or moral principles. Indeed, abortion politics has become so entangled in considerations of any form of human embryo research that bioethicist R. Alta Charo of the University of Wisconsin-Madison told the NAS panel that "the United States is almost incapable of a sensible policy discussion in this area."

Still, in the next few weeks, the NAS panel plans to release recommendations to guide legislators as they consider regulation of human cloning. A moratorium or an outright ban on human cloning seems likely, and several countries have already asked the United Nations to pass such a restriction.

RELATED ARTICLE: Are cloned monkeys next?

The birth of the sheep named Dolly provoked an international furor about the possibility of human cloning. Don Wolf worries that the world will similarly overreact if he and his colleagues clone an adult nonhuman primate, such as a rhesus monkey.

That fear "is a disincentive to continue, but [we] simply can't be intimidated," he says. "There's such a need for genetically identical monkeys, such as for AIDS-vaccine work, that we need to press on."

Several years ago, Wolf's team at the Oregon Regional Primate Research Center in Beaverton successfully cloned monkeys by using the nucleus of an embryonic cell (SN: 3/8/97, p. 142). Yet it hasn't succeeded when starting with the nucleus of a cell from an adult monkey. Indeed, the researchers are still struggling to get such cloned embryos ready for transfer into a surrogate mother.

"Progress has been slow and has been limited. We're trying to establish conditions where we can get [cloned] embryos to grow to the implantation stage. Once we do that with a reasonable degree of regularity, we'll go back to doing embryo transfers to establish pregnancies," Wolf told SCIENCE NEWS. "We're on the cusp." --J.T.