Japanese Biologist Cloned A Mouse And Cloned The Clone

Creating exact copies or doppelgängers of creatures in a lab was largely the stuff of science fiction until 1996, when Dolly the Sheep became the first mammal ever to be cloned. But while speculations arose about the potential pitfalls of generating hordes of clones, as a team of Japanese researchers found out, those issues sometimes take on a different form than expected.

Two decades ago, Teruhiko Wakayama (a biologist from the University of Yamanashi) and his team of researchers cloned a single female mouse. Then, they cloned the clone, and the clone’s clone.

With initially promising results, they continued to clone from each successive generation of clones for a total of 58 generations (not including the original mouse). But problems began to rear their heads after generation 25—when re-clones from one of the later generations mated with male mice, egg fertilization succeeded, but embryos degenerated. It turned out that, despite having no abnormalities in appearance or lifespan, more and more mutations had begun to accumulate in the DNA of each successive generation. Eventually, the combined effects of the mutations became lethal.

Creatures that clone themselves repeatedly do exist in nature, so it’s not impossible to do. Hydra and coral polyps are just two examples of animals that can self-replicate, but as it turns out, mammals (which rely on sexual reproduction) are a different story. For many years, it was thought that cloning could potentially be used to produce superior domesticated animals en masse, or bring endangered species back from the brink. But after seeing what happened with the 1,200 mice they cloned, the researchers behind the 20-year experiment aren’t so sure.

"[Mammal cloning] issues are believed to stem from failures in "reprogramming" the donor nucleus to reset the epigenetic state of differentiated somatic cells to that of a fertilized embryo; this occurs not directly due to DNA abnormalities, but the detailed mechanisms responsible remain unclear," they said in a study recently published in the journal Nature Communications.

Given that genetic abnormalities don’t appear in successive generations of cloned plants, non-mammalian vertebrates cloned through parthenogenesis, or less complex cloning animals (such as hydras), researchers wanted to see if mammals could also be cloned indefinitely.

The first rounds of results looked hopeful — after serial cloning trails began in 2005, DNA sequencing and analysis of duplicated mice revealed no differences between early and later generations of clones. But that began to change after generation 27, when genetic abnormalities began to negatively affect fertility. By the 58th generation, the cloned mice died the day after birth.

There may have been several causes behind the abnormalities that arose in later generations. One suspect was Trichostatin A (TSA), which enhances nuclear reprogramming—a process that resets cells to have the differentiation abilities of pluripotent stem cells without changing any of their nuclear DNA. While the researchers suspected that TSA lost its effectiveness as generations progressed, analysis showed otherwise.

Epigenetic abnormalities—changes to gene expression that switch genes on and off without affecting the structure of the DNA sequence — were another possibility, but no such abnormalities were found. Embryos of mice that were cloned in vitro also showed nothing abnormal in their development.

It was only when the researchers sequenced the genomes of re-cloned mice from different generations that they found mutations. Disastrously, by the final generation, so many background mutations had accumulated that the clones were no longer viable. While many re-cloned mice with multiple mutations were able to live out healthy lives (and some even bred with mice that were not clones), successive cloning ultimately backfired. These results raise serious doubts about whether cloning can provide a way to save endangered mammal species.

"Since the birth of Dolly the sheep, cloning technology has been envisioned for diverse applications," the researchers said. "Yet, these findings demonstrate that practical application of cloning of mammals requires a deeper appreciation of these biological constraints, warranting further research, and reaffirm the evolutionary inevitability that sexual reproduction is indispensable for the long-term survival of mammalian species."