title: Remodeling somatic nuclei via exogenous expression of protamine 1 to create spermatid-like structures for somatic nuclear transfer.
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.
authors.affiliation: Faculty of Veterinary Medicine, University of Teramo, Teramo, Italy.
authors.forename: Marta
authors.initials: M
authors.lastname: Czernik
authors.affiliation: Faculty of Veterinary Medicine, University of Teramo, Teramo, Italy.
authors.forename: Domenico
authors.initials: D
authors.lastname: Iuso
authors.affiliation: Faculty of Veterinary Medicine, University of Teramo, Teramo, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Toschi
authors.affiliation: INSERM, U823, Institut Albert Bonniot, Université Grenoble Alpes, Grenoble, France.
authors.forename: Saadi
authors.initials: S
authors.lastname: Khochbin
authors.affiliation: Faculty of Veterinary Medicine, University of Teramo, Teramo, Italy.
authors.forename: Pasqualino
authors.initials: P
authors.lastname: Loi
mesh_terms: D000818:Animals; D015870:Gene Expression; D008297:Male; D051379:Mice; D041681:NIH 3T3 Cells; D053652:Nuclear Transfer Techniques; D010957:Plasmids / Q000235:genetics; D011479:Protamines / Q000235:genetics*; D012756:Sheep; D013087:Spermatids / Q000166:cytology*
chemical_list: D011479:Protamines