Production of healthy cloned mice from bodies frozen at −20°C for 16 years

Abstract

Cloning animals by nuclear transfer provides an opportunity to preserve endangered mammalian species. However, it has been suggested that the ''resurrection'' of frozen extinct species (such as the woolly mammoth) is impracticable, as no live cells are available, and the genomic material that remains is inevitably degraded. Here we report production of cloned mice from bodies kept frozen at ؊20°C for up to 16 years without any cryoprotection. As all of the cells were ruptured after thawing, we used a modified cloning method and examined nuclei from several organs for use in nuclear transfer attempts. Using brain nuclei as nuclear donors, we established embryonic stem cell lines from the cloned embryos. Healthy cloned mice were then produced from these nuclear transferred embryonic stem cells by serial nuclear transfer. Thus, nuclear transfer techniques could be used to ''resurrect'' animals or maintain valuable genomic stocks from tissues frozen for prolonged periods without any cryopreservation.brain ͉ cloning ͉ cryopreservation ͉ nuclear transfer ͉ reprogramming C urrent animal cloning techniques using nuclear transfer (1) can reconstruct animals from donor cells; clones so produced differ only in their mitochondrial DNA (2). This technology has produced a variety of cloned animals for scientific and commercial purposes. However, it is not known whether this method could be applied to the recovery of animals from frozen extinct species. Although most, if not all, mammalian cells lose their functional integrity if frozen without cryoprotective reagents, it is conceivable that some aspects of genomic information remain intact even in such dead bodies. By contrast, the genomes of spermatozoa are resistant to freezing (3), and even freeze-drying (4) without cryoprotectants. However, it has been difficult to demonstrate this in somatic cells, due to technical difficulties in assessing genomic integrity.One recent paper has reported on the nuclear integrity of dead cells after freeze-thawing by improved techniques (5). This study successfully produced cloned mouse embryos and generated germ line chimerae via nuclear transferred embryonic stem cell (ntES) cells. However, although all donor cells were dead after thawing, the donor cells were frozen in a medium including polyvinylpyrrolidone, which is known to exhibit cryoprotectant effect (6). Freeze-dried cells were also used for these nuclear transfer attempt, and all cells were dead after rehydration (7,8). Although cloned embryos were obtained from those dead cells following nuclear transfer, no cloned offspring were obtained. Moreover, so far all studies of nuclear transfer using ''dead'' material have used isolated single cells frozen in the laboratory using special media. In dead specimens frozen in natural conditions such as permafrost tundra, the cells of tissue will presumably bind strongly to each other and freeze gradually after death due to the large body size. It remains to be shown whether nuclei can be collected from whole bodies frozen without cryopro...

Nuclear Transfer Preserves the Nuclear Genome of Freeze-Dried Mouse Cells

“…We have already succeeded in producing live cloned mice from mouse somatic cells frozen for 16 years [7], and this suggests that the viability of donor cells is not important for producing the next generation using nuclear transfer. Recently, Loi et al reported that sheep blastocysts could be generated from freeze-dried somatic cells with a similar success rate as for fresh cells following storage at room temperature for 3 years [8].…”

Germ cells and fertilization: why I studied these topics and what I learned along the path of my study

“…This male mouse, died of an unexpected flood, was the sole animal with unique genotype. To make this story short, we could not clone this mouse because that time we did not know how to recover and handle cells from frozen body for cloning, which can be performed today (Wakayama et al, 2008). Nevertheless, cloning by somatic nuclear transfer should be a temporal, supplemental method for animal reproduction.…”

Improvement of mouse cloning using nuclear transfer-derived embryonic stem cells and/or histone deacetylase inhibitor

“…Even in infertile and 'unclonable' strains of mice, we can generate offspring from somatic cells by combining cloning with ntES technology Wakayama et al, 2005a;Wakayama et al, 2005b). Moreover, cloned offspring can be generated potentially even from the nuclei of dead bodies or cells Wakayama et al, 2008), such as from frozen extinct animals. Currently, only the ntES technology is available for this purpose, because all other techniques, including induced pluripotent stem (iPS) cell derivation, require significant numbers of living donor cells.…”