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

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Abstract

This protocol describes 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, we also provide step-by-step instructions for somatic cell nuclear transfer (SCNT) 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.

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Figure 1: Protaminized adult fibroblasts in sheep and mouse.
Figure 2: Schematic diagram of the protaminization of somatic cell nuclei, isolation and use of donors for the somatic cell nuclear transfer (SCNT) procedure.
Figure 3: Visualization of protaminized nuclei of fresh somatic cells using a Nikon Eclipse TE 300 fluorescence microscope.
Figure 4: Piezo micro-tool fabrication.
Figure 5: Suggested layout of droplets in the manipulation chambers of the 10-cm dish.
Figure 6: Primary cells isolated from adult ear tissue.
Figure 7: Visualization of protaminized nuclei of fixed somatic cells under a fluorescence microscope.
Figure 8: Fully protaminized somatic nucleus ideal for nuclear transfer (NT).
Figure 9: Microscopy images of sheep oocytes.
Figure 10: Enucleation of sheep oocytes and injection of protaminized somatic nucleus using the Piezo unit.
Figure 11: Gradual protamine incorporation into the somatic nucleus.
Figure 12: Photographic (top) and schematic (bottom) representation of spermatic and protaminized nuclei.

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Acknowledgements

This work was funded by grant MIUR/CNR, Program FIRB GA B81J12002520001 (GenHome), and the European Union's Horizon2020 Project "ERAofART" (GA 692185), to P.L. The authors are participating in the COST action FA 1201 'Epiconcept' Epigenetic and Peri-conception Environment. The authors warmly acknowledge L. Palazzese for help with the photos. The authors acknowledge FlowMetric Europe SpA (Italy), an affiliate company of FlowMetric (USA), for cell sorting.

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Author notes

  1. Marta Czernik and Domenico Iuso: These authors contributed equally to this work.

Authors and Affiliations

  1. Faculty of Veterinary Medicine, University of Teramo, Teramo, Italy

    Marta Czernik, Domenico Iuso, Paola Toschi & Pasqualino Loi

  2. INSERM, U823, Institut Albert Bonniot, Université Grenoble Alpes, Grenoble, France

    Saadi Khochbin

Authors
  1. Marta Czernik
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  2. Domenico Iuso
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  3. Paola Toschi
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  4. Saadi Khochbin
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  5. Pasqualino Loi
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Contributions

M.C., D.I., P.T., S.K. and P.L. contributed to the development of the methodology and to the description of the protocol; M.C. and D.I. performed the experiments and prepared the figures. M.C., D.I., S.K. and P.L. wrote the manuscript.

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Correspondence to Pasqualino Loi.

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The authors declare no competing financial interests.

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Czernik, M., Iuso, D., Toschi, P. et al. Remodeling somatic nuclei via exogenous expression of protamine 1 to create spermatid-like structures for somatic nuclear transfer. Nat Protoc 11, 2170–2188 (2016). https://doi.org/10.1038/nprot.2016.130

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