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      Analyzing bovine OCT4 and NANOG enhancer activity in pluripotent stem cells using fluorescent protein reporters

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          Abstract

          Green fluorescent protein (GFP) reporters controlled by the regulatory region of OCT4 and NANOG—two master regulators for pluripotency are widely used in studies of pluripotent stem cell establishment and embryo development. Alongside the challenge in establishing bovine pluripotent stem cells, the application of bovine-specific gene reporters has rarely been explored.

          Using lentivirus-based GFP reporter, we investigated the upstream regulatory regions of bovine OCT4 and NANOG. These reporters show activity in both naïve- and primed-state pluripotency when infected into mouse and human embryonic stem cells (ESCs), respectively. Consistent with what is found in humans and mice, the bovine OCT4-distal enhancer (b OCT4-DE) but not the proximal enhancer (b OCT4-PE) region is preferentially activated in naïve-state pluripotency. Furthermore, the b OCT4-DE region is silenced upon conversion of naive-state ESCs into primed-state epiblast stem cells (EpiSCs). Co-infection of mouse fibroblasts with the reprograming factors for induced pluripotent stem cell (iPSC) induction leads to the generation of GFP positive colonies, demonstrating that these GFP reporters can serve as live indicators for induced pluripotent cell establishment. We further proved that the bovine OCT4 distal enhancer is active in bovine blastocysts. We established the lentiviral-based fluorescent reporters controlled by bovine OCT4 and NANOG enhancer sequences. These reporter constructs show activity in naïve- and primed-pluripotent states. These reporters may serve as versatile tools for bovine ESC/iPSC generation and identification, as well as for developmental studies of bovine embryos.

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          Most cited references27

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          Naive and primed pluripotent states.

          After maternal predetermination gives way to zygotic regulation, a ground state is established within the mammalian embryo. This tabula rasa for embryogenesis is present only transiently in the preimplantation epiblast. Here, we consider how unrestricted cells are first generated and then prepared for lineage commitment. We propose that two phases of pluripotency can be defined: naive and primed. This distinction extends to pluripotent stem cells derived from embryos or by molecular reprogramming ex vivo.
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            Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4.

            Oct4 is a mammalian POU transcription factor expressed by early embryo cells and germ cells. We report that the activity of Oct4 is essential for the identity of the pluripotential founder cell population in the mammalian embryo. Oct4-deficient embryos develop to the blastocyst stage, but the inner cell mass cells are not pluripotent. Instead, they are restricted to differentiation along the extraembryonic trophoblast lineage. Furthermore, in the absence of a true inner cell mass, trophoblast proliferation is not maintained in Oct4-/- embryos. Expansion of trophoblast precursors is restored, however, by an Oct4 target gene product, fibroblast growth factor-4. Therefore, Oct4 also determines paracrine growth factor signaling from stem cells to the trophectoderm.
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              Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3.

              The propagation of embryonic stem (ES) cells in an undifferentiated pluripotent state is dependent on leukemia inhibitory factor (LIF) or related cytokines. These factors act through receptor complexes containing the signal transducer gp130. The downstream mechanisms that lead to ES cell self-renewal have not been delineated, however. In this study, chimeric receptors were introduced into ES cells. Biochemical and functional studies of transfected cells demonstrated a requirement for engagement and activation of the latent trancription factor STAT3. Detailed mutational analyses unexpectedly revealed that the four STAT3 docking sites in gp130 are not functionally equivalent. The role of STAT3 was then investigated using the dominant interfering mutant, STAT3F. ES cells that expressed this molecule constitutively could not be isolated. An episomal supertransfection strategy was therefore used to enable the consequences of STAT3F expression to be examined. In addition, an inducible STAT3F transgene was generated. In both cases, expression of STAT3F in ES cells growing in the presence of LIF specifically abrogated self-renewal and promoted differentiation. These complementary approaches establish that STAT3 plays a central role in the maintenance of the pluripotential stem cell phenotype. This contrasts with the involvement of STAT3 in the induction of differentiation in somatic cell types. Cell type-specific interpretation of STAT3 activation thus appears to be pivotal to the diverse developmental effects of the LIF family of cytokines. Identification of STAT3 as a key transcriptional determinant of ES cell self-renewal represents a first step in the molecular characterization of pluripotency.
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                Author and article information

                Contributors
                Role: Formal analysisRole: Funding acquisitionRole: InvestigationRole: ValidationRole: Writing – original draft
                Role: Formal analysisRole: InvestigationRole: MethodologyRole: ValidationRole: VisualizationRole: Writing – original draft
                Role: ResourcesRole: ValidationRole: VisualizationRole: Writing – original draft
                Role: Formal analysisRole: Validation
                Role: ConceptualizationRole: InvestigationRole: MethodologyRole: Writing – review & editing
                Role: ConceptualizationRole: InvestigationRole: MethodologyRole: Writing – review & editing
                Role: ConceptualizationRole: ResourcesRole: Writing – review & editing
                Role: Funding acquisitionRole: InvestigationRole: Writing – original draft
                Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                5 October 2018
                2018
                : 13
                : 10
                : e0203923
                Affiliations
                [1 ] State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Animal Reproduction Institute, Guangxi University, Nanning, Guangxi, China
                [2 ] Department of Animal Science, Institute for Systems Genomics, University of Connecticut, Storrs, CT, United States of America
                [3 ] U.S. Department of Agriculture, Agricultural Research Service, Animal Biosciences and Biotechnology Laboratory, Beltsville, MD, United States of America
                The Roslin Institute, UNITED KINGDOM
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0001-6188-7758
                Article
                PONE-D-17-39139
                10.1371/journal.pone.0203923
                6173392
                30289916
                f0dfc89d-a1a7-46b9-9794-bd1c682c06cb

                This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

                History
                : 3 November 2017
                : 30 August 2018
                Page count
                Figures: 5, Tables: 0, Pages: 15
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100005825, National Institute of Food and Agriculture;
                Award ID: 2016-67016-24894
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100005825, National Institute of Food and Agriculture;
                Award ID: USDA W2171 regional project
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100005240, Changjiang Scholar Program of Chinese Ministry of Education;
                Award ID: Chinese Scholarship Council for Study Abroad
                Award Recipient :
                This work was supported by the United States Department of Agriculture (USDA) W2171 regional project to Y.T., the Agriculture and Food Research Initiative Competitive Grant no. 2016-67016-24894 to Y.T. from the USDA National Institute of Food and Agriculture ( https://nifa.usda.gov/), and the Chinese Scholarship Council for Study Abroad for D.H. and M.Z. ( https://www.csc.edu.cn/chuguo).
                Categories
                Research Article
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Stem Cells
                Cell Potency
                Pluripotency
                Biology and Life Sciences
                Developmental Biology
                Cell Differentiation
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Stem Cells
                Induced Pluripotent Stem Cells
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Stem Cells
                Research and Analysis Methods
                Spectrum Analysis Techniques
                Spectrophotometry
                Cytophotometry
                Flow Cytometry
                Biology and Life Sciences
                Developmental Biology
                Embryology
                Blastocysts
                Research and Analysis Methods
                Microscopy
                Light Microscopy
                Fluorescence Microscopy
                Biology and Life Sciences
                Biochemistry
                Proteins
                Luminescent Proteins
                Green Fluorescent Protein
                Custom metadata
                All relevant data are within the paper and its Supporting Information files.

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