This is what happened when a scientist made a clone of a clone of a clone of a clone

In 2005, Teruhiko Wakayama, a pioneer of cloning at the University of Yamanashi in Japan, cloned a single female mouse. The results were as expected: The mouse that was born was genetically identical to the female they started with. But then, Wakayama decided to do something extraordinary. He and his team began creating a theoretically infinite chain of re-cloning. That female cloned mouse was cloned. And then, that cloned mouse was cloned again. 

As of this past year, that mouse has been re-cloned over 1,200 times. 

“Copying a picture on a photocopier results in slightly lower image quality. Copying that copy again makes the quality even worse,” says Wakayama. “We wanted to see what would happen if the same thing occurred with a cloned mouse.”

Now, results published in Nature Communications are helping scientists understand the hard limits of this technology, which is becoming an essential tool in conservation and agriculture, and what it means for rescuing species from the brink of extinction. As it turns out, yes, a clone of a clone of a clone is indeed a blurry copy. 

How to clone a clone

Over 20 years, Wakayama and his team attempted to clone more than 30,000 times, successfully cloning around 1,200 mice, with up to four generations born each year. They produced 58 generations total, using a cloning technique known as somatic cell nuclear transfer, which extracts the nucleus from a donor cell and injects it into a cell without a nucleus known as a denucleated egg, producing multiple cloned embryos. 

At first, the success rate—as measured by the number of mice born relative to the number of cloned embryos transferred to surrogates—seemed to increase generation after generation, climbing from 7 percent in the beginning to 15.5 percent in the 26^th generation. But then something unexpected happened, Wakayama says. From the 27^th generation on, the success rate started declining, reaching 0.6 percent in the 57^th and 58^th generations. 

To find out why the success rate was plummeting, the researchers sequenced the genome of mice from different generations and found that during each re-cloning, harmful genetic mutations were building up—Wakayama says that previous studies had implied most clones would be free of mutations. By the 27^th generation, the process had reached a turning point, causing a steady decrease. 

The results show that when cloning a mammal, scientists aren’t necessarily creating a perfect copy of the original. “We were really surprised because we thought the clone was exactly the same as the original donor,” says Wakayama. In fact, cloned mice developed three times as many mutations as ordinary mice, he notes.

The mutations could be caused by the absence of sexual reproduction, which naturally limits the buildup of harmful mutations, but it might also arise from the cloning process itself.

“The fact that re-cloned animals would have more mutations than naturally bred organisms is absolutely expected,” says Ben Novak, a specialist in wildlife conservation biotechnologies at Revive & Restore, which funds research to apply biotechnologies to conservation challenges. “The clones are being re-cloned from somatic tissue, and we know that somatic tissue, over the lifetime of an organism, accumulates mutations.… But it's just really cool to finally see a paper that analyzed that kind of data, especially across so many generations.”

With a little help from cloning

Many mammals have been cloned since Dolly the sheep was born in 1996, from cats and dogs to horses and pigs, and some experts believe that mammal cloning might be the key to solving some of life’s problems. 

For example, by cloning specific livestock, farmers can “copy” the most fertile members of their herds or those most resistant to diseases. In polo, enthusiasts have cloned the best-performing horses, and pets are being cloned too. But there are controversies around those practices; for example, some critics argue that cloning pets is unethical, given the large number of shelter animals euthanized each year.

On the other hand, cloning could help conservation by boosting genetic diversity, which can be low in small, endangered populations and is often a critical problem for a species’ survival. Biobanks all over the world store genetic material as a lifeline that can be used to help restore or revive species.

For example, researchers are cloning black-footed ferrets using the genetic material of a ferret who died about 30 years ago. They hope the clone’s descendants, once reintroduced in the wild, will boost genetic diversity of the endangered species.

And if humans ever migrate to other planets, cloning could come in handy, says Wakayama, since you could avoid transporting large animals through space and instead clone their genome.

“Cloning is its own industry already,” says Katarzyna Malin, a developmental endocrinologist in the VetART Lab at UC Davis, who was not involved in Wakayama’s research. “It’s more that the ethical concerns are nuanced, and application needs to be responsible and with sophistication.”  

She says the new research should urge prevention-based conservation efforts, in lieu of relying on cloning right now. “Optimistically, serial cloning may one day offer a gateway to maintain breeding of a specific animal for periods or a few lifespans,” she explains. “This study emphasizes, however, that contrary to the researchers’ expectations, serial cloning does not work indefinitely.”

Even though re-cloning is not a common practice, Novak says the study helps advance scientists’ knowledge of a technology still in its early stages. 

“I think studying clones like this is actually really powerful and important for the future of figuring out more about how genomes work and what's the tolerance with how many mutations you can accumulate before it's bad,” Novak says. “If we have studies like this…then we can have real, grounded conversations, rather than speculation.”