Remodeling somatic nuclei via exogenous expression of protamine 1 to create spermatid-like structures for somatic nuclear transfer

Developers

Marta Czernik, Domenico Iuso, Paola Toschi, Saadi Khochbin, Pasqualino Loi et al.

Description of the technology

This technology provides a protocol, describing how to convert the chromatin structure of sheep and mouse somatic cells into spermatid-like nuclei through the heterologous expression of the protamine 1 gene (Prm1). Furthermore, the protocol also provides step-by-step instructions for somatic cell nuclear transfer of Prm1-remodeled somatic nuclei in sheep oocytes. There is evidence that changing the organization of a somatic cell nucleus with that which mirrors the spermatozoon nucleus leads to better nuclear reprogramming.

The protocol may have further potential application in determining the protamine and histone footprints of the whole genome; obtaining 'gametes' from somatic cells; and furthering understanding of the molecular mechanisms regulating the maintenance of DNA methylation in imprinted control regions during male gametogenesis. The protocol is straightforward, and it requires 4 weeks from the establishment of the cell lines to their transfection and the production of cloned blastocysts. It is necessary for researchers to have experience in cell biology and embryology, with basic skills in molecular biology, to carry out the protocol.

Practical application

The protocol is promising for somatic cell nuclear transfer. Embryonic development has statistically higher percentages of successful implementation up to the blastocyst stage when ‘protaminized’ cells, as compared with control, untransfected fibroblasts (14% versus 7%), were used for somatic cell nuclear transfer. In addition to that, the protocol might be useful for scientists interested in understanding the following biological issues:

1. Use of this technology as a simplified model for whole-genome protaminization.
2. Determination of the protamine/histone footprints on the whole genome.
3. The protocol could improve understanding of the molecular mechanisms regulating the maintenance of DNA methylation in imprinted control regions during male gametogenesis. The whole-genome protaminization protocol will allow researchers to monitor the maintenance/removal of imprinted marks during nuclear compaction.

The main advantage of somatic cell protaminization technique is that is straightforward, fast (only 48 h for full protaminization of the nucleus) and highly efficient. Moreover, no advanced instruments or particular molecular biology skills are required to carry out the protocol.

Laboratories

• Faculty of Veterinary Medicine, University of Teramo, Teramo (Italy)
• INSERM, U823, Institut Albert Bonniot, Université Grenoble Alpes, Grenoble (France)

Links

Publications

• Czernik, M. et al. «Remodeling somatic nuclei via exogenous expression of protamine 1 to create spermatid-like structures for somatic nuclear transfer." 11 Nature Protocols (2016): 2170–2188.
• Loi, P., Iuso, D., Czernik, M. & Ogura, A. «A new, dynamic era for somatic cell nuclear transfer?» Trends Biotechnol. Apr 22. pii: S0167-7799(16)30003–8 (2016).
• Ogura, A. et al. «Recent advancements in cloning by somatic cell nuclear transfer." 368 Philos. Trans. R. Soc. Lond. B Biol. Sci. (2013): 20110329.