19 Exploring the expression of protamine 1 as a “xeroprotectant” in somatic cell nuclear transfer using sheep freeze-dried adult fibroblast

Somatic cell nuclear transfer (SCNT) is the only nuclear reprogramming method that allows the rewinding of an adult nucleus into a totipotent stage. As such, it offers excellent opportunities for the multiplication of elite genotypes or endangered animals, whose numbers have shrunk to below the threshold of safe existence on this planet. Disappointingly, SCNT efficiency is still unreliable; hence, it would be wise to store somatic cells from threatened animals in biobanks. We were the first to show that freeze-dried somatic cells could be reactivated to generate blastocyst-stage embryos upon nuclear transfer. Only a few papers have been published on the topic since then, and recently, by Wakayama and colleagues, the first offspring was obtained in the mouse model, but with a double nuclear transfer technique. On the other hand, lyophilisation of mammalian spermatozoa has made considerable progress, partially thanks to the physical stability that the protamine(s) confer on the DNA. In our previous work, we have demonstrated that a somatic cell could be made more amenable to the oocyte reprogramming tools by the exogenous expression of human protamine 1. Given that the protamine also provides protection against dehydration stress, we have combined our cell protaminisation breakthrough with our cell and gamete lyophilisation expertise. Briefly, adult sheep fibroblasts were transfected with plasmid containing the RFP-tagged mouse protamine 1 gene (pmPrm1-RFP) using Lipofectamine 2000 (ThermoFisher). At 48 h after transfection, the efficiency was assessed by reposting a positive number of cells as 40% on total number of cells. The Prm1-cells were collected and centrifuged with Ficoll gradient in order to enrich the number of Prm1-cells. Subsequently, the Prm1-cells were suspended in the freeze-dry solution, frozen, and dried with a normal bench-top freeze-drier. Freeze-dried Prm1-cells were used as donor nuclei in enucleated sheep oocytes and embryonic development followed. Currently, only one test with a limited number of reconstructed embryos has been performed. We are confident that our contribution will be relevant for establishing easily reprogrammable somatic cell collections stored at low cost in the dry form in biobanks for endangered species.

This work has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under the Marie Sklodowska-Curie grant agreement No. 734434. Moreover, the present study was carried out in the framework of the Project ‘Demetra’ (Dipartimenti di Eccellenza 2018–2022, CUP_C46C18000530001), funded by the Italian Ministry for Education, University and Research. L.P. and M.C. acknowledge support from the National Science Centre, Poland, through grant No. 2016/21/D/NZ3/02610 (Sonata) and 2019/35/B/NZ3/02856 (Opus).