Institute of Genetics and Animal Biotechnology of the Polish Academy of Sciences, Jastrzebiec, Poland.
Faculty of Veterinary Medicine, University of Teramo, Teramo, Italy.
Methods Mol Biol. 2023;2647:211-224. doi: 10.1007/978-1-0716-3064-8_11.
Somatic cell nuclear transfer (SCNT) is the only nuclear reprogramming method that allows rewinding an adult nucleus into a totipotent state. As such, it offers excellent opportunities for the multiplication of elite genotypes or endangered animals, whose number have shrunk to below the threshold of safe existence. Disappointingly, SCNT efficiency is still low. Hence, it would be wise to store somatic cells from threatened animals in biobanks. We were the first to show that freeze-dried cells allow generating blastocysts upon SCNT. Only a few papers have been published on the topic since then, and viable offspring have not been produced. On the other hand, lyophilization of mammalian spermatozoa has made considerable progress, partially due to the physical stability that protamines provide to the genome. In our previous work, we have demonstrated that a somatic cell could be made more amenable to the oocyte reprogramming by the exogenous expression of human Protamine 1. Given that the protamine also provides natural protection against dehydration stress, we have combined the cell protaminization and lyophilization protocols. This chapter comprehensively describes the protocol for somatic cell protaminization, lyophilization, and its application in SCNT. We are confident that our protocol will be relevant for establishing somatic cells stocks amenable to reprogramming at low cost.
体细胞核移植（Somatic Cell Nuclear Transfer，SCNT）是唯一一种能够使成年细胞核重编程为全能状态的核重编程方法。因此，它为精英基因型或濒危动物的繁殖提供了极好的机会，这些动物的数量已经减少到低于安全生存的阈值。令人失望的是，SCNT 的效率仍然很低。因此，明智的做法是将受威胁动物的体细胞储存在生物库中。我们是第一个证明冻干细胞可以通过 SCNT 产生囊胚的人。从那时起，只有少数几篇关于这个主题的论文发表，而且没有产生可育的后代。另一方面，哺乳动物精子的冷冻干燥已经取得了相当大的进展，部分原因是鱼精蛋白为基因组提供了物理稳定性。在我们之前的工作中，我们已经证明，通过外源表达人源 Protamine 1，可以使体细胞更易于接受卵母细胞重编程。鉴于鱼精蛋白还能提供天然的抗脱水应激保护，我们将细胞鱼精蛋白化和冻干方案结合起来。本章全面描述了体细胞鱼精蛋白化、冻干及其在 SCNT 中的应用的方案。我们相信，我们的方案将有助于以低成本建立可重编程的体细胞库。
