Daiyu Ito

Abstract Mammals can now be cloned artificially, but it remains unknown whether they can also maintain their species through cloning. Herein, we continued serial cloning for 20 years from a single donor mouse. These re-cloned mice appeared normal and had normal lifespans, but large structural and lethal mutations accumulated in their DNA with each generation. The birth rate of serial cloning began to decline from the 27th generation, and the 58th generation was the last.

Animals BDF1 (C57BL/6N × DBA/2), BCF1 (C57BL/6N × C3H/He), 129B6F1-GFPTg (129/Sv  × C57BL/6N-GFP), BD129F1 (BDF1 × 129/Sv), C3H/He, C57BL/6N, and ICR mice (8–10 weeks of age) were obtained from SLC Inc. (Hamamatsu, Japan) or produced in our mouse facility. Surrogate pseudo-pregnant ICR females, which were used as embryo recipients, were mated with vasectomised ICR males, the sterility of which had been demonstrated previously.

Highlights • Successful preservation of mouse freeze-dried (FD) spermatozoa by thin plastic sheet • DNA integrity and developmental potential are comparable to the conventional method • Viable mouse FD sperm can be transferred via an air mail without any protection • Tens of thousands of mouse strains can be preserved in a single “sperm-book” Summary Freeze-drying techniques allow the preservation of mammalian spermatozoa without using liquid nitrogen.