Resurrection of a Bull by Cloning from Organs Frozen without Cryoprotectant in a −80°C Freezer for a Decade

Abstract

Frozen animal tissues without cryoprotectant have been thought to be inappropriate for use as a nuclear donor for somatic cell nuclear transfer (SCNT). We report the cloning of a bull using cells retrieved from testicles that had been taken from a dead animal and frozen without cryoprotectant in a −80°C freezer for 10 years. We obtained live cells from defrosted pieces of the spermatic cords of frozen testicles. The cells proliferated actively in culture and were apparently normal. We transferred 16 SCNT embryos from these cells into 16 synchronized recipient animals. We obtained five pregnancies and four cloned calves developed to term. Our results indicate that complete genome sets are maintained in mammalian organs even after long-term frozen-storage without cryoprotectant, and that live clones can be produced from the recovered cells.

In vitro culture of fibroblast-like cells from postmortem skin of Katahdin sheep stored at 4°C for different time intervals

“…Integrity of nuclear genome of donor cells is essential for full-term development of cloned animals (Hoshino et al 2009). The quality of donor cells including the viability, proliferation rate, longevity, and normality in culture ensures genomic integrity and enhances the success rate of animal cloning (Mastromonaco et al 2006).…”

“…The quality of donor cells including the viability, proliferation rate, longevity, and normality in culture ensures genomic integrity and enhances the success rate of animal cloning (Mastromonaco et al 2006). In vitro culture of cells from live or dead animal tissues preserved at sub-zero temperatures with or without cryoprotectants has been achieved (Wakayama et al 2008;Hoshino et al 2009;Erker et al 2010). The nucleus from these cultured cells from preserved tissues has been used to clone the animals even after many years of their death (Wakayama et al 2008;Hoshino et al 2009).…”

Genome Banking for Vertebrates Wildlife Conservation

“…Somatic cells from a wide variety of sources can be used for this purpose. Such diverse sources include cells from tissues preserved without cryoprotectant at -80ºC for more than a decade, or cells from tissues kept at -20ºC for as long as 16 years (Hoshino et al, 2009), cells isolated from mummified animals (Kato et al, 2009), freeze-dried somatic cells (Loi et al, 2008a;Ono et al, 2008; see next section), semen-derived somatic cells (Nel-Themaat et al, 2008a;Nel-Themaat et al, 2008b;Liu et al, 2010), cells collected postmortem (Oh et al, 2008), cell line (Campbell et al, 1996), and of course both fetal and adult cells are suitable for this purpose (Wilmut et al, 1997). SCNT has indeed an obvious potential for the multiplication of rare genotypes (Corley- Smith & Brandhorst, 1999;Loi et al, 2008a;Loi et al, 2008b), but its wide application is prevented by the currently low efficiency in terms of offspring outcome.…”

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

“…Cloning animals by SCNT provides an opportunity to preserve endangered mammalian species as when viable cells can be collected from frozen bodies, it is possible to generate cloned animals (Hoshino et al, 2009). However, the 'resurrection' of extinct species from permafrost (such as the woolly mammoth) is thought to be impractical, because no live cells will be available.…”