For all of her six years, Dolly the sheep was a symbol of success -- living proof that mammals could indeed be cloned.
But her relatively early death -- prompted Friday by a progressive lung infection, which followed bouts with arthritis and obesity -- is sure to be a sobering reminder for cloning researchers of how complex and poorly understood the process remains.
It may never be known whether Dolly’s maladies had anything to do with her unnatural start in life.
Cloning is a field in its infancy, a craft characterized more by failure than success. In animal experiments, barely one in 100 cloned embryos survives to birth. Of those survivors, many have health problems such as heart, lung and weight abnormalities.
A growing number of studies suggest that clones -- even the ones that make it to adulthood -- may carry within them subtle genetic abnormalities that could cause medical problems later in life.
Scientists say it is crucial that these subtleties be carefully explored and that many cloned animals’ lives be monitored until death, so as to properly understand the scope and consequences of such problems. They say the findings underscore how irresponsible and dangerous it would be to try to clone human beings given the current state of knowledge.
Beyond the moral and ethics debates about cloning -- and the likelihood that, so far, the only claims of cloned babies have been elaborate hoaxes -- researchers say it will probably be years, if not decades, before science understands how to clone a human without introducing minuscule genetic errors that could create deformed and diseased children.
“I think it’s very likely that every single clone will have something wrong with it,” said Davor Solter, director of the Max-Planck Institute of Immunobiology in Freiburg, Germany. “We are stuck with these problems today -- but it doesn’t mean we will never know how to solve them.”
Cloning has long seemed to hover on the edge of possibility -- ripe for any outlandish claim -- partly because the concept is tantalizingly simple. By replacing the genetic material of a fertilized egg with that of a body cell, you could, in theory, create an exact copy of another living organism.
Achieving this goal was another matter. It was only in the 1950s and 1960s that scientists successfully cloned frogs; mammals proved tougher.
In 1981, scientists reported they had cloned three mice -- but no one could repeat the experiments, and some still suspect the claim was fraudulent. For a long while after, many believed that cloning mammals was impossible.
At last came the spectacular 1997 announcement of Dolly’s birth -- and, soon after, of Cumulina the cloned mouse (named for the “cumulus cell” from which she was made), followed by cloned cows, goats, pigs, rabbits and one small kitten called Carbon Copy.
Success, however, comes hard. To get Dolly, it took 277 tries; Carbon Copy the kitten came after 86 failures.
Mark Westhusin, associate professor of veterinary medicine at Texas A&M; University, estimates that a scientist starting with 100 cow eggs might get 60 to 70 of them to survive to the one-cell embryo stage, and 20 to the 100-cell embryo stage. Sixteen cows might still be pregnant 35 days after implantation, and one or two calves might be born alive.
About one-fourth of those would have obvious, serious deformities or health problems, and some would die.
Those that live may do fine. Advanced Cell Technology Inc., a Worcester, Mass., company researching cloning, has found that of 30 successfully cloned cows, 80% of them were alive and apparently healthy at ages 1 to 5 years, although more recently one of the cows developed a benign tumor and another epileptic seizures. Hundreds of other cloned animals are also alive and appear healthy.
However, one cannot perform subtle behavioral tests on a cow or sheep to scan for errors of mind and mood -- the kinds of problems that might not matter as much in a cow but would be problematic in a child.
“For the life of me, I don’t know if Dolly was autistic or schizophrenic -- I don’t know how you assay that in a sheep,” said Gerald Schatten, a University of Pittsburgh professor and director of the Pittsburgh Development Center, a biology research institute.
One also cannot know whether clones have natural life spans because few have lived long enough yet.
However, a group headed by Rudolf Jaenisch, scientist at the Whitehead Institute for Biomedical Research in Cambridge, Mass., has examined 10,000 mouse genes in the livers and placentas of cloned mice and found that hundreds of the genes exhibited abnormal patterns of activity. And Japanese researchers have found that cloned mice died earlier than normal mice, dying prematurely of pneumonia, damaged livers and tumors.
“Even if clones appear normal at a younger age, they are not,” Jaenisch said. “You just have to wait.”
Even the most basic steps in the process remain fiddly and delicate, akin to performing brain surgery with hammers, chisels and cattle prods.
The process begins by drilling into the egg, removing its genetic material then replacing it with DNA from a body cell, such as a skin or mammary cell.
The next step is to trick the egg into thinking it’s a bona fide fertilized egg, so that it starts dividing -- a feat achieved with electrical or chemical triggers.
Many clones fail at these early stages simply because of physical damage to the nucleus or the cell’s viscous innards, which are stuffed with chemicals essential for normal development.
But some of the most serious potential problems -- miscarriages and birth defects -- run deeper than mere manhandling. Embryo growth is a delicate chemical dance, filled with subtle cascades of gene and protein activity that are blunted, augmented or mistimed during cloning.
Normally, embryo division is activated by precise changes in calcium levels, choreographed by a chemical, oscillin, which the sperm brings into the egg.
In cloning, the timing of those changes is off, and the result can be errors and birth defects. Normally, eggs and early embryos are nurtured in the fallopian tubes and uterus. But in cloning, they are cultured in dishes until they’re ready to be implanted. Studies show that embryo culture can cause errors even if the embryo isn’t a clone.
Scientists are also beginning to understand that cloned and in vitro embryos have different needs. For example, Keith Latham, an associate professor in the Fels Institute for Cancer Research and Molecular Biology at Temple University in Philadelphia, has found that mouse clones thrive when fed glucose, while normal mouse embryos prefer other kinds of sugars.
Many scientists believe that the most critical problem with cloning lies in the unnatural origin of a clone’s genetic material.
Normally, a fertilized egg contains genetic material from sperm and egg. The genes in these two cells are turned on or off in a precise pattern that is in perfect readiness for the complex development of an embryo. But the genes of the body cells used in cloning are not in that state of readiness. When they are added to an egg, the egg tries its best to reprogram them, but it wasn’t built to do this job and it doesn’t do it very well.
“Skin cells, or whatever scientists use for cloning, are in a totally different state,” Jaenisch said. “No wonder it goes wrong. I am surprised that it works even partially.”