An orientation experiment reveals a major unexpected problem with cloning

A clone is supposed to be a genetically identical copy, but an extraordinary 20-year study has shown that this is not actually the case. He reveals that the clones have a lot of other mutations, and if you keep cloning the clones, they will grow to lethal levels. The findings have implications for the use of cloning in agriculture and for the rescue of endangered animals, including efforts to recreate extinct species, as well as the potential use of cloning technology in humans.

The big question is why there are so many mutations in the clones. It could be that adult body cells that are cloned accumulate more mutations than egg or sperm cells. But Teruhiko Wakayama at Yamanashi University in Japan, thinks that at least some of them may be caused by the cloning process itself. “Unfortunately, while the clones were once thought to be identical to the original, they have turned out not to be, suggesting that there may be problems with their use,” he says. “In the future, we have to prove that mutations arising from cloning do not pose problems.”

Cloning mammals was once thought impossible because as cells in the body develop and specialize, various chemical tags are added or removed to parts of the genome that control gene activity. The DNA of the skin cells, shall we say, is “programmed” to make skin cells. But the birth of Dolly the sheep in July 1996 showed that transferring the nucleus of an adult cell into an empty egg could reprogram its genome and allow the egg to develop. Soon after, Wakayama created Cumulina, the first cloned mouse, born in October 1997.

To test how well his team’s mouse cloning method worked, Wakayama began cloning clones in 2005. “Just as copying a painting results in a lower quality image, I wanted to check how the clones compared to the original,” he says.

In 2013, he and his colleagues reported that they had repeatedly cloned clones for 25 consecutive generations, generating more than 500 mice from the original donor. “The cloned mice produced in our experiments showed no physical abnormalities in any generation, lived as long as normal mice, and were healthy,” says Wakayama.

However, this success has not been achieved in other species – there are still high rates of health problems in cloned dogs, and no one has yet cloned a primate from an adult cell. But in mice, Wakayama thought that repeated cloning could go on indefinitely. However, as his team continued to experiment, the success rate decreased until finally, in the 58th generation, none of the clones survived.

To find out why, the team now sequenced the genomes of 10 mice from different generations. This revealed that there were an average of more than 70 mutations per clone generation – three times as many as in a control group of mice that reproduced naturally. In particular, large mutations began to accumulate in cloned mice after the 27th generation, eventually losing the entire X chromosome.

The explanation could simply be that animals have developed ways to protect sperm and egg cells from mutations and to remove harmful mutations during sexual reproduction, meaning that adult body cells end up with many more mutations. For example, a recent study found that mutations accumulated eight times faster in blood cells compared to sperm. So if the adult cells that are cloned have more mutations to begin with, so will the clones.

But Wakayama thinks that some other mutations are caused by the nuclear transfer process itself. “It’s not surprising that the nucleus—that is, the DNA—can be damaged by physical shock,” he says. “I believe that if we could develop a more subtle method of nuclear transfer, we might be able to reduce the mutation rate in cloned embryos. However, I have no ideas yet on how to achieve this.”

Shukrat Mitalipov at Oregon Health & Science University is skeptical. “Any observed increase in mutation rate in clones is more likely to reflect the genomic state of the donor cells, rather than a uniform effect of the nuclear transfer process itself,” he says.

While human cloning is banned in many countries, researchers like Mitalipov are exploring the use of nuclear transfer to create matching tissues or organs for medical treatment and to generate sperm and eggs to treat infertility. Wakayama’s results show the importance of careful selection of donor cells and screening when that happens, Mitalipov says. “Ideally, the donor cell population should be screened for deleterious variants. Where necessary, gene-editing approaches could be used to correct known deleterious mutations.”

But if the cloning process itself induces mutations, that would not be enough. To be clear, these findings do not mean that using cloning techniques is too risky—the mutation rate per generation is still relatively low, and cells can be tested for dangerous mutations after cloning—but they do show that there are even more potential problems than we thought. An already problematic technology just got worse.