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      Mitochondrial DNA Depletion in Granulosa Cell Derived Nuclear Transfer Tissues

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          Abstract

          Somatic cell nuclear transfer (SCNT) is a key technology with broad applications that range from production of cloned farm animals to derivation of patient-matched stem cells or production of humanized animal organs for xenotransplantation. However, effects of aberrant epigenetic reprogramming on gene expression compromise cell and organ phenotype, resulting in low success rate of SCNT. Standard SCNT procedures include enucleation of recipient oocytes before the nuclear donor cell is introduced. Enucleation removes not only the spindle apparatus and chromosomes of the oocyte but also the perinuclear, mitochondria rich, ooplasm. Here, we use a Bos taurus SCNT model with in vitro fertilized (IVF) and in vivo conceived controls to demonstrate a ∼50% reduction in mitochondrial DNA (mtDNA) in the liver and skeletal muscle, but not the brain, of SCNT fetuses at day 80 of gestation. In the muscle, we also observed significantly reduced transcript abundances of mtDNA-encoded subunits of the respiratory chain. Importantly, mtDNA content and mtDNA transcript abundances correlate with hepatomegaly and muscle hypertrophy of SCNT fetuses. Expression of selected nuclear-encoded genes pivotal for mtDNA replication was similar to controls, arguing against an indirect epigenetic nuclear reprogramming effect on mtDNA amount. We conclude that mtDNA depletion is a major signature of perturbations after SCNT. We further propose that mitochondrial perturbation in interaction with incomplete nuclear reprogramming drives abnormal epigenetic features and correlated phenotypes, a concept supported by previously reported effects of mtDNA depletion on the epigenome and the pleiotropic phenotypic effects of mtDNA depletion in humans. This provides a novel perspective on the reprogramming process and opens new avenues to improve SCNT protocols for healthy embryo and tissue development.

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          Most cited references 91

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          Viable offspring derived from fetal and adult mammalian cells.

          Fertilization of mammalian eggs is followed by successive cell divisions and progressive differentiation, first into the early embryo and subsequently into all of the cell types that make up the adult animal. Transfer of a single nucleus at a specific stage of development, to an enucleated unfertilized egg, provided an opportunity to investigate whether cellular differentiation to that stage involved irreversible genetic modification. The first offspring to develop from a differentiated cell were born after nuclear transfer from an embryo-derived cell line that had been induced to become quiescent. Using the same procedure, we now report the birth of live lambs from three new cell populations established from adult mammary gland, fetus and embryo. The fact that a lamb was derived from an adult cell confirms that differentiation of that cell did not involve the irreversible modification of genetic material required for development to term. The birth of lambs from differentiated fetal and adult cells also reinforces previous speculation that by inducing donor cells to become quiescent it will be possible to obtain normal development from a wide variety of differentiated cells.
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            Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction

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              Sheep cloned by nuclear transfer from a cultured cell line.

              Nuclear transfer has been used in mammals as both a valuable tool in embryological studies and as a method for the multiplication of 'elite' embryos. Offspring have only been reported when early embryos, or embryo-derived cells during primary culture, were used as nuclear donors. Here we provide the first report, to our knowledge, of live mammalian offspring following nuclear transfer from an established cell line. Lambs were born after cells derived from sheep embryos, which had been cultured for 6 to 13 passages, were induced to quiesce by serum starvation before transfer of their nuclei into enucleated oocytes. Induction of quiescence in the donor cells may modify the donor chromatin structure to help nuclear reprogramming and allow development. This approach will provide the same powerful opportunities for analysis and modification of gene function in livestock species that are available in the mouse through the use of embryonic stem cells.
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                Author and article information

                Contributors
                Journal
                Front Cell Dev Biol
                Front Cell Dev Biol
                Front. Cell Dev. Biol.
                Frontiers in Cell and Developmental Biology
                Frontiers Media S.A.
                2296-634X
                14 May 2021
                2021
                : 9
                Affiliations
                1Department of Veterinary Medicine, University of Sassari , Sassari, Italy
                2Molecular Animal Breeding and Biotechnology, Gene Center and Department of Veterinary Science, LMU Munich , Munich, Germany
                3ETH Zürich, Animal Physiology, Institute of Agricultural Sciences , Zurich, Switzerland
                4Bavarian State Research Center for Agriculture, Institute of Animal Breeding , Grub, Germany
                5Bayern-Genetik GmbH , Grub, Germany
                6Livestock Sciences, South Australian Research and Development Institute , Roseworthy, SA, Australia
                7School of Animal and Veterinary Sciences, The University of Adelaide , Roseworthy, SA, Australia
                8Davies Research Centre, School of Animal and Veterinary Sciences, The University of Adelaide , Roseworthy, SA, Australia
                9Robinson Research Institute, The University of Adelaide , Adelaide, SA, Australia
                Author notes

                Edited by: Robert Feil, UMR 5535 Institut de Génétique Moléculaire de Montpellier (IGMM), France

                Reviewed by: Kumiko Takeda, National Agriculture and Food Research Organization (NARO), Japan; Marta Czernik, University of Teramo, Italy; Nathalie Beaujean, Le Nouvel Institut National de Recherche sur l’Agriculture, l’Alimentation et l’Environnement en France INRAE, France

                *Correspondence: Daniela Bebbere, dbebbere@ 123456uniss.it

                This article was submitted to Developmental Epigenetics, a section of the journal Frontiers in Cell and Developmental Biology

                Article
                10.3389/fcell.2021.664099
                8194821
                Copyright © 2021 Bebbere, Ulbrich, Giller, Zakhartchenko, Reichenbach, Reichenbach, Verma, Wolf, Ledda and Hiendleder.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                Page count
                Figures: 5, Tables: 0, Equations: 3, References: 91, Pages: 13, Words: 0
                Funding
                Funded by: Deutsche Forschungsgemeinschaft 10.13039/501100001659
                Categories
                Cell and Developmental Biology
                Original Research

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