Abstract
The unprecedented decline of biodiversity worldwide is urging scientists to collect and store biological material from seriously threatened animals, including large mammals. Lyophilization is being explored as a low-cost system for storage in bio-banks of cells that might be used to expand or restore endangered or extinct species through the procedure of Somatic Cell Nuclear Transfer (SCNT). Here we report that the genome is intact in about 60% of lyophylized sheep lymphocytes, whereas DNA damage occurs randomly in the remaining 40%. Remarkably, lyophilized nuclei injected into enucleated oocytes are repaired by a robust DNA repairing activity of the oocytes, and show normal developmental competence. Cloned embryos derived from lyophylized cells exhibited chromosome and cellular composition comparable to those of embryos derived from fresh donor cells. These findings support the feasibility of lyophylization as a storage procedure of mammalian cells to be used for SCNT.
Highlights
The biological activity of some organisms can be reversibly suspended when intracellular water is either unavailable following exposure to freezing temperatures, or lost as a result of severe water deprivation
The capacity to survive dehydration is conferred by the disaccharide trehalose, a sugar that starts to accumulate in cells once water stress is sensed
Freeze-dried lymphocytes are devoid of water The water activity of fresh blood lymphocytes suspended in freezing solution was 0.996, while the resulting freeze-dried lymphocytes were devoid of free water, with aw of 0.224
Summary
The biological activity of some organisms can be reversibly suspended when intracellular water is either unavailable following exposure to freezing temperatures, or lost as a result of severe water deprivation. Tardigrades (Milnesium tardigradum) and sleeping chironomids (Polypedilum vanderplanki) are the most studied and best examples [6], but the list of desiccation-tolerant organisms encompasses four phyla [7]. The capacity to survive dehydration is conferred by the disaccharide trehalose, a sugar that starts to accumulate in cells once water stress is sensed. Late Embryogenesis Abundant (LEA) proteins, which were originally identified in seeds [8], play a role in desiccation-tolerance [7]. Sugars and LEA proteins exercise their protective effect against desiccation by binding to lipid membranes and macromolecules to form a glassy weft that reversibly holds their interaction [9]
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