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

• Published in: PloS one Vol. 4; no. 1; p. e4142
• Main Authors: Hoshino, Yoichiro, Hayashi, Noboru, Taniguchi, Shunji, Kobayashi, Naohiko, Sakai, Kenji, Otani, Tsuyoshi, Iritani, Akira, Saeki, Kazuhiro
• Format: Journal Article
• Language: English
• Published: San Francisco Public Library of Science 08.01.2009
• Subjects: Animal tissues; Animals; Biotechnology; Bos gaurus; Bos javanicus; bulls; Calves; Cattle; Cell culture; Cloning; cloning (animals); Cords; cryopreservation; Cryoprotectants; Cryoprotectors; Developmental Biology/Cell Differentiation; Developmental Biology/Embryology; embryo transfer; embryogenesis; Embryos; Endangered & extinct species; Experiments; Extinction; Genetic engineering; Genomes; Genomics; Livestock; Nuclear transfer; nuclear transplantation; Organs; Paternity; pregnancy; Rodents; Somatic cell nuclear transfer; spermatic cord; testes; Japan
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0004142

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.