Landmark experiment reveals a big 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 the case. He reveals that the clones have many additional mutations, and if you continue to clone clones, these reach lethal levels. The findings have implications for the use of cloning in agriculture and for saving endangered animals, including efforts to recreate extinct species, as well as for the potential use of cloning technology in humans.

The big question is why there are so many mutations in the clones. It could simply be that adult body cells that are cloned accumulate more mutations than eggs or sperm. But Teruhiko Wakayama, of Yamanashi University in Japan, thinks the cloning process itself could be the cause of at least some of them. “Unfortunately, while it was once thought that clones were identical to the original, it has become clear that this is not the case, suggesting that there may be problems with their use,” he says. “In the future, we need to demonstrate that mutations resulting from cloning do not cause problems. »

Cloning mammals was once thought impossible because, as the body’s cells grow and specialize, various chemical tags that control gene activity are added or removed from parts of the genome. The DNA in skin cells, for example, is “programmed” to make skin cells. But the birth of the sheep Dolly 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. Shortly after, Wakayama created Cumulina, the first cloned mouse, born in October 1997.

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

In 2013, he and his colleagues announced that they had repeatedly cloned clones over 25 successive 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.

This success has not been achieved with other species, however: there is still a high rate of health problems in cloned dogs and no one has yet cloned a primate from an adult cell. But in mice, Wakayama believed that repeated cloning could continue indefinitely. Yet as his team continued their experiments, the success rate dropped until by the 58th generation, none of the clones had survived.

To find out why, the team sequenced the genomes of 10 mice from different generations. This revealed that there were on average more than 70 mutations per generation of clones, three times more than in a control group of mice that reproduced naturally. In particular, large-scale mutations began to develop in cloned mice after the 27th generation, eventually leading to the loss of an entire X chromosome.

The explanation could simply be that animals have evolved ways to protect sperm and eggs from mutations and eliminate harmful mutations during sexual reproduction, meaning that cells in the adult body end up with many more mutations. For example, a recent study found that mutations accumulate eight times faster in blood cells than in sperm. So if the cloned adult cells have more mutations, the clones will have them too.

But Wakayama thinks the nuclear transfer process itself is causing some additional mutations. “It is not surprising that the nucleus, that is, the DNA, is damaged by the physical shock,” he says. “I think that if we could develop a gentler nuclear transfer method, we might be able to reduce the mutation rate in cloned embryos. However, I don’t have any ideas yet on how to achieve this.”

Shoukhrat Mitalipov of Oregon Health & Science University is skeptical. “Any observed increase in mutation rates 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 such as Mitalipov are exploring the use of nuclear transfer to generate matching tissues or organs for medical treatments, as well as to generate sperm and eggs to treat infertility. Wakayama’s results show the importance of careful selection and screening of donor cells if this is done, says Mitalipov. “Ideally, donor cell populations should be assessed for deleterious variants. If necessary, gene editing approaches could be used to correct known harmful mutations.”

But if the cloning process itself induces mutations, that won’t be enough. To be clear, these results don’t mean that cloning techniques are too risky to use – the mutation rate per generation is still relatively low and cells can be screened after cloning to check for dangerous mutations – but they do show that there are even more potential problems than we thought. An already problematic technology becomes even more so.