Clones produced from mice frozen for 16 years

With current technology, could we clone a mammoth? Cloning techniques have made significant progress in recent years and at least one well-preserved specimen has been found. But the same freezing process that preserves the bodies of many extinct mammals would also be the undoing of cloning endeavours. Ice destroys cells, puncturing their membranes, bursting them and exposing their contents. Upon thawing, the dead cells would be useless as a basis for cloning.

Until now, the destructive power of ice seemed like an insurmountable obstacle, dashing the prospect of "resurrecting" frozen extinct animals or preserving endangered ones. But a Japanese team have made a technical breakthrough that could both into more realistic visions. They have managed to breed healthy cloned mice from thawed bodies that have been frozen for 16 years. It's quite literally a small start, but an important one.

A typical procedure for cloning is as follows. Take a living cell from a donor animal and fuse it with an 'empty' egg cell that lacks a nucleus - the central part of the cell that contains the DNA. The result is an egg that contains the donor's genetic information, which can then be implanted into a surrogate mother. For this recipe, thawed cells from frozen tissues make poor ingredients; with their shredded membranes, they cannot fuse with other cells.

Sayaka Wakayama and colleagues from RIKEN in Japan solved the problem by developing a way of directly injecting the nucleus of a donor cell into the empty egg, broken membranes be damned. At first, they tested their method using cells taken from mice that had been frozen at -20C for a week. Even though the thawed bodies were bereft of living cells, the technique worked and while the success rate was low, there were successes nonetheless.

Some organs worked better than others; blood proved to be a reasonable source of donor material but the brain was even better. With its thawed nuclei, Wakayama managed to produce cloned embryos with a success rate of 39%, an efficiency that rivals that of fresh tissue.

Wakayama thinks that the brain is such a suitable organ for the technique because it requires a lot of sugar to work properly. And some sugars, such as sucrose and trehalose, can protect cells from the damaging effects of freezing. Alternatively, the same icy process that breaks the membranes of cells and renders them useless for standard cloning methods might have spelled success for Wakayama's technique. Perhaps, by puncturing the membranes, the ice crystals cleared a path to the nucleus for the various reprogramming chemicals that are used in cloning.

Whatever the reason, the group's technique clearly worked. With it, they managed to produce living cloned mice using nuclei extracted from the brains of three individuals that had been on ice for up to a month. They even managed to clone frozen mice by first using nuclei from their brain and blood cells to produce lines of stem cells. The method may be more indirect, but Wakayama says that it's actually easier, and it allowed them to create clones from mice that had been frozen for a much longer span of 16 years.

Their work greatly surpasses previous attempts. Another group of scientists almost managed to clone mice by using cells that had been thawed after freezing but their attempt was only partially successful - it produced embryos, but none of them survived till birth. And the cells used were isolated and specially frozen in the laboratory, using chemicals to protect them from the harsher aspects of the freezing process. Such controlled conditions would hardly apply to an ancient animal that froze in natural conditions or an endangered one deliberately chilled in the field without access to sophisticated facilities.

Wakayama's methods are better matched to such natural conditions. There are still many technical hurdles to clear before the practical applications of this technique become valid. The need for a surrogate mother is an obvious one, especially for extinct species. What, for example, would be a suitable host for a mammoth? An elephant? This new paper doesn't mean that we've crossed the finish line by any means, but it does put us back in the race.

A quick note: I usually frown on science journalism that puts the "practical application" of a piece of research before the discussion of the research itself. But in this case, I think it's a valid approach - the practical side isn't a reflection of a throwaway line at the end of the paper; it's a core motivating factor in the research and discussed frequently throughout the paper.

Reference: PNAS doi:S. Wakayama, H. Ohta, T. Hikichi, E. Mizutani, T. Iwaki, O. Kanagawa, T. Wakayama (2008). Production of healthy cloned mice from bodies frozen at -20 C for 16 years Proceedings of the National Academy of Sciences, 105 (45), 17318-17322 DOI: 10.1073/pnas.0806166105