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 trasfer (SCNT). We report the cloning of a bull using cells retrieved rom testicles that had ben 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 trasferred 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 recovered cells. © 2009 Hoshino et al.