62 RECOVERY OF CELL NUCLEI FROM 15 000-YEAR-OLD MAMMOTH TISSUES AND INJECTION INTO MOUSE ENUCLEATED MATURED OOCYTES

Since the report of “Dolly” (Wilmut et al. 1997 Nature 385, 810-813), cloned animals have been successfully produced by somatic cell nuclear transfer (SCNT). In those reports, cultured or fresh cells were mainly used as nuclear donor cells in SCNT. Meanwhile, there was a report of the birth of offspring by SCNT with the nuclear donor cell derived from frozen whole body kept at -20°C for 16 years without cryoprotectant (Wakayama et al. 2008 Proc. Natl. Acad. Sci. USA 105, 17 318-17 322). This report suggests that if tissues or cells are cryopreserved postmortem without cryoprotectant, resurrection of the postmortem animal individual might be possible by SCNT. Furthermore, in cases of extinct or endangered species, it is difficult to use oocytes derived from same species as the recipient cytoplasm. However, there was a report of the birth of offspring by SCNT with oocytes derived from closely related species (Lanza et al. 2000 Cloning 2, 79-90). This result suggests that if oocytes from closely related species are available as the recipient cytoplasm, it would be possible to resurrect extinct or endangered species by SCNT. Woolly mammoth (Mammuthus primigenius) is a famous animal that existed during the ice ages and became extinct at the end of the last ice age. Its relics have been excavated from permafrost. Recently, the prospect of the resurrection of woolly mammoth by SCNT has heightened. However, even if the soft tissue of the woolly mammoth is excavated, it is not clear whether its cell nuclei retain their biological characteristics for more than several thousand years. In this study, we recovered cell nuclei from 15 000-year-old mammoth tissues and injected them into mouse enucleated matured oocytes as the nuclear donor in SCNT. Frozen mammoth legs were excavated from Northeast Siberian permafrost at the point of 71°34′56.9″ N and 141°37′37.6″ E. The conventional radiocarbon age of the sample after 13C correction was 13 100 to 12800 BC or 12400 to 11 800 BC. Cell nucleus-like structures were successfully recovered from skin and muscle tissues. Those nuclei were then injected into enucleated BDF1 mouse oocytes, and more than half of the injected oocytes were able to survive (67%; 12/18 for oocytes injected with skin-derived nucleus, 55%; 72/131 for oocytes injected with muscle-derived nucleus and 33%; 26/80 for oocytes injected with frozen-thawed mouse bone marrow-derived nucleus as the control group). Injected nuclei were not taken apart and retained their nuclear structure. Those oocytes did not show disappearance of nuclear membrane or premature chromosome condensation at 1 h after injection and did not form pronuclear-like structures at 7 h after injection. Because one-half of the oocytes injected with nuclei derived from frozen-thawed mouse bone marrow cells were able to form pronuclear-like structures (46%; 12/26), it might be possible to promote the cell cycle of nuclei from ancient animal tissues by suitable pretreatment in SCNT. This is the first report of SCNT with nuclei derived from mammoth tissues. This study was funded in part by Gifu Prefecture, Japan.