Mouse frozen for 16 years and its clone. (A) C3H/He strain male mouse frozen at −20 °C for 16 years. (B and C), Frozen brain tissues were collected on dry ice. (D and E) Donor nuclei were collected from a brain fragment by gentle homogenization. (E) Nuclei were stained with Hoechst 33342. (F) A cloned mouse derived from C3H/He strain male mouse frozen at −20 °C for 16 years via secondary nuclear transfer from an ntES cell. One clone died from respiratory failure just after birth and one clone showed open eyelids at birth but was cannibalized the next day by the foster mother. However, the remaining two clones grew to adulthood. (G) The first C3H/He strain cloned mouse (agouti) has demonstrated normal fertility (all offspring were agouti) when mated with a female (black). The female clone was derived from BDF1 frozen brain via ntES cell nuclear transfer. (H) Although the rate of establishment of ntES cell lines decreased with the increased storage period, the sample frozen for 16 years retained the potential to establish ntES cell lines after nuclear transfer. Blue bar, derived from brain nuclei; green bar, derived from tail blood; red bar, derived from C3H strain brain nuclei.