Cloning: Back from the ice

Many scientists have considered using cloning to resurrect extinct animals frozen in permafrost, such as the woolly mammoth. This has proven to be difficult because there are no live cells, and DNA is irreparably damaged by ice crystals.

Now cloning techniques may have progressed to the point where resurrection from permafrost could become reality. Teruhiko Wakayama at the RIKEN Center for Developmental Biology in Kobe and co-workers have produced healthy cloned mice from cells that were frozen for 16 years at -20 °C without any preserving chemicals—simulating the conditions in permafrost (1).

Wakayama and his team adapted a common cloning technique called somatic cell nuclear transfer (SCNT). Usually, researchers collect a single live cell of the donor animal and fuse it with a living egg that has had its own nucleus removed. However dead cells cannot be used for cell fusion, so Wakayama invented a new injection method in which the nuclei from dead cells are directly injected into the egg.

The researchers first performed SCNT on cells from mice frozen for a week. They found that frozen brain cells were the best donor nuclei and, surprisingly, the frozen cell nuclei yielded more healthy clones than living brain cells. It is possible that all the sugars in the brain protect cells from freezing damage. Furthermore, the freezing process might partly unravel the tight bundle of nuclear DNA, allowing the host egg to access the donor’s genetic code more easily.

The normal SCNT procedure failed to produce cloned mice from bodies frozen for 16 years, but the researchers managed to establish embryonic stem cell lines. The stem cell nuclei were injected into other eggs and transferred into surrogate mothers, leading to four healthy cloned mice.

This work represents the first successful cloning from bodies stored in conditions similar to the natural permafrost environment. Wakayama is hopeful that they could eventually produce clones from ancient bodies of extinct species such as mammoths.

“Cells frozen in permafrost for thousands of years are not only frozen but also dried out,” he explains. “This means there is probably more damage in the nuclei than in our 16-year-frozen cells. However, we already published results this year in which we succeeded in using freeze-dried cells for nuclear transfer (2).

“What’s more, our paper demonstrated that even blood cells can be used as nuclear donors. Blood cells are found in any tissue, including skin and bones, which are most likely to be found in permafrost.”

1. Wakayama, S., Ohta, H., Hikichi, T., Mizutani, E., Iwaki, T., Kanagawa, O. & Wakayama, T. Production of healthy cloned mice from bodies frozen at -20 °C for 16 years. Proceedings of the National Academy of Sciences 105, 17318–17322 (2008).

2. Ono, T., Mizutani, E., Li, C. & Wakayama, T. Nuclear transfer preserves the nuclear genome of freeze-dried mouse cells. The Journal of Reproduction and Development In press. (2008)

The corresponding author for this highlight is based at the RIKEN Laboratory for Genomic Reprogramming