Somatic Cell Nuclear Transfer Using Freeze-Dried Protaminized Donor Nuclei.

ABSTRACT

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.