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  • 1
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    SC3-seq: a method for highly parallel and quantitative measurement of single-cell gene expression (2015)
    Oxford University Press
    Details
    Publication Date: 2015-05-20
    Description: Single-cell mRNA sequencing (RNA-seq) methods have undergone rapid development in recent years, and transcriptome analysis of relevant cell populations at single-cell resolution has become a key research area of biomedical sciences. We here present s ingle- c ell mRNA 3 -prime end seq uencing (SC3-seq), a practical methodology based on PCR amplification followed by 3-prime-end enrichment for highly quantitative, parallel and cost-effective measurement of gene expression in single cells. The SC3-seq allows excellent quantitative measurement of mRNAs ranging from the 10,000-cell to 1-cell level, and accordingly, allows an accurate estimate of the transcript levels by a regression of the read counts of spike-in RNAs with defined copy numbers. The SC3-seq has clear advantages over other typical single-cell RNA-seq methodologies for the quantitative measurement of transcript levels and at a sequence depth required for the saturation of transcript detection. The SC3-seq distinguishes four distinct cell types in the peri-implantation mouse blastocysts. Furthermore, the SC3-seq reveals the heterogeneity in human-induced pluripotent stem cells (hiPSCs) cultured under on-feeder as well as feeder-free conditions, demonstrating a more homogeneous property of the feeder-free hiPSCs. We propose that SC3-seq might be used as a powerful strategy for single-cell transcriptome analysis in a broad range of investigations in biomedical sciences.
    Keywords: Massively Parallel (Deep) Sequencing
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 2
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    Meikin is a conserved regulator of meiosis-I-specific kinetochore function (2014)
    Nature Publishing Group (NPG)
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    Publication Date: 2014-12-24
    Description: The kinetochore is the crucial apparatus regulating chromosome segregation in mitosis and meiosis. Particularly in meiosis I, unlike in mitosis, sister kinetochores are captured by microtubules emanating from the same spindle pole (mono-orientation) and centromeric cohesion mediated by cohesin is protected in the following anaphase. Although meiotic kinetochore factors have been identified only in budding and fission yeasts, these molecules and their functions are thought to have diverged earlier. Therefore, a conserved mechanism for meiotic kinetochore regulation remains elusive. Here we have identified in mouse a meiosis-specific kinetochore factor that we termed MEIKIN, which functions in meiosis I but not in meiosis II or mitosis. MEIKIN plays a crucial role in both mono-orientation and centromeric cohesion protection, partly by stabilizing the localization of the cohesin protector shugoshin. These functions are mediated mainly by the activity of Polo-like kinase PLK1, which is enriched to kinetochores in a MEIKIN-dependent manner. Our integrative analysis indicates that the long-awaited key regulator of meiotic kinetochore function is Meikin, which is conserved from yeasts to humans.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Kim, Jihye -- Ishiguro, Kei-ichiro -- Nambu, Aya -- Akiyoshi, Bungo -- Yokobayashi, Shihori -- Kagami, Ayano -- Ishiguro, Tadashi -- Pendas, Alberto M -- Takeda, Naoki -- Sakakibara, Yogo -- Kitajima, Tomoya S -- Tanno, Yuji -- Sakuno, Takeshi -- Watanabe, Yoshinori -- England -- Nature. 2015 Jan 22;517(7535):466-71. doi: 10.1038/nature14097. Epub 2014 Dec 24.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Laboratory of Chromosome Dynamics, Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1Yayoi, Tokyo 113-0032, Japan. ; Instituto de Biologia Molecular y Celular del Cancer (CSIC-USAL), 37007 Salamanca, Spain. ; Center for Animal Resources and Development, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811 Japan. ; Laboratory for Chromosome Segregation, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/25533956" target="_blank"〉PubMed〈/a〉
    Keywords: Animals ; Cell Cycle Proteins/metabolism ; Centromere/metabolism ; Chromosomal Proteins, Non-Histone/deficiency/genetics/*metabolism ; *Conserved Sequence ; Female ; Humans ; Infertility/genetics/metabolism ; Kinetochores/*metabolism ; Male ; *Meiosis ; Mice ; Molecular Sequence Data ; Protein-Serine-Threonine Kinases/metabolism ; Proto-Oncogene Proteins/metabolism ; Saccharomyces cerevisiae Proteins/metabolism ; Schizosaccharomyces pombe Proteins/metabolism
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
    Published by Nature Publishing Group (NPG)
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  • 3
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    Distinct cohesin complexes organize meiotic chromosome domains (2003)
    American Association for the Advancement of Science (AAAS)
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    Publication Date: 2003-05-17
    Description: Meiotic cohesin complexes at centromeres behave differently from those along chromosome arms, but the basis for these differences has remained elusive. The fission yeast cohesin molecule Rec8 largely replaces its mitotic counterpart, Rad21/Scc1, along the entire chromosome during meiosis. Here we show that Rec8 complexes along chromosome arms contain Rec11, whereas those in the vicinity of centromeres have a different partner subunit, Psc3. The arm associated Rec8-Rec11 complexes are critical for meiotic recombination. The Rec8-Psc3 complexes comprise two different types of assemblies. First, pericentromeric Rec8-Psc3 complexes depend on histone methylation-directed heterochromatin for their localization and are required for cohesion during meiosis II. Second, central core Rec8-Psc3 complexes form independently of heterochromatin and are presumably required for establishing monopolar attachment at meiosis I. These findings define distinct modes of assembly and functions for cohesin complexes at different regions along chromosomes.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Kitajima, Tomoya S -- Yokobayashi, Shihori -- Yamamoto, Masayuki -- Watanabe, Yoshinori -- New York, N.Y. -- Science. 2003 May 16;300(5622):1152-5.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/12750522" target="_blank"〉PubMed〈/a〉
    Keywords: Cell Division ; Centromere/physiology ; Chromatids/*physiology ; Chromatin ; *Chromosome Segregation ; Chromosomes, Fungal/*physiology ; Green Fluorescent Proteins ; Luminescent Proteins ; Meiosis/*physiology ; Phosphoproteins/*physiology ; Schizosaccharomyces ; Schizosaccharomyces pombe Proteins/genetics/*physiology
    Print ISSN: 0036-8075
    Electronic ISSN: 1095-9203
    Topics: Biology , Chemistry and Pharmacology , Computer Science , Medicine , Natural Sciences in General , Physics
    Published by American Association for the Advancement of Science (AAAS)
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  • 4
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    PRC1 coordinates timing of sexual differentiation of female primordial germ cells (2013)
    Nature Publishing Group (NPG)
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    Publication Date: 2013-03-15
    Description: In mammals, sex differentiation of primordial germ cells (PGCs) is determined by extrinsic cues from the environment. In mouse female PGCs, expression of stimulated by retinoic acid gene 8 (Stra8) and meiosis are induced in response to retinoic acid provided from the mesonephroi. Given the widespread role of retinoic acid signalling during development, the molecular mechanisms that enable PGCs to express Stra8 and enter meiosis in a timely manner are unknown. Here we identify gene-dosage-dependent roles in PGC development for Ring1 and Rnf2, two central components of the Polycomb repressive complex 1 (PRC1). Both paralogues are essential for PGC development between days 10.5 and 11.5 of gestation. Rnf2 is subsequently required in female PGCs to maintain high levels of Oct4 (also known as Pou5f1) and Nanog expression, and to prevent premature induction of meiotic gene expression and entry into meiotic prophase. Chemical inhibition of retinoic acid signalling partially suppresses precocious Oct4 downregulation and Stra8 activation in Rnf2-deficient female PGCs. Chromatin immunoprecipitation analyses show that Stra8 is a direct target of PRC1 and PRC2 in PGCs. These data demonstrate the importance of PRC1 gene dosage in PGC development and in coordinating the timing of sex differentiation of female PGCs by antagonizing extrinsic retinoic acid signalling.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Yokobayashi, Shihori -- Liang, Ching-Yeu -- Kohler, Hubertus -- Nestorov, Peter -- Liu, Zichuan -- Vidal, Miguel -- van Lohuizen, Maarten -- Roloff, Tim C -- Peters, Antoine H F M -- England -- Nature. 2013 Mar 14;495(7440):236-40. doi: 10.1038/nature11918.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH-4058 Basel, Switzerland.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/23486062" target="_blank"〉PubMed〈/a〉
    Keywords: Adaptor Proteins, Signal Transducing ; Animals ; Chromatin/genetics/metabolism ; Down-Regulation ; Female ; Gene Expression Regulation, Developmental ; Homeodomain Proteins/metabolism ; Male ; Meiosis ; Mice ; Octamer Transcription Factor-3/genetics/metabolism ; Ovum/*cytology/*metabolism ; Polycomb Repressive Complex 1/deficiency/*metabolism ; Polycomb Repressive Complex 2/metabolism ; Proteins/genetics ; Sex Characteristics ; Sex Differentiation/*physiology ; Signal Transduction ; Time Factors ; Transcription, Genetic ; Tretinoin/metabolism ; Ubiquitin-Protein Ligases/deficiency/metabolism
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
    Published by Nature Publishing Group (NPG)
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  • 5
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    Generation of human oogonia from induced pluripotent stem cells in vitro (2018)
    American Association for the Advancement of Science (AAAS)
    In: Science
    Details
    Publication Date: 2018
    Description: 〈p〉Human in vitro gametogenesis may transform reproductive medicine. Human pluripotent stem cells (hPSCs) have been induced into primordial germ cell–like cells (hPGCLCs); however, further differentiation to a mature germ cell has not been achieved. Here, we show that hPGCLCs differentiate progressively into oogonia-like cells during a long-term in vitro culture (approximately 4 months) in xenogeneic reconstituted ovaries with mouse embryonic ovarian somatic cells. The hPGCLC-derived oogonia display hallmarks of epigenetic reprogramming—genome-wide DNA demethylation, imprint erasure, and extinguishment of aberrant DNA methylation in hPSCs—and acquire an immediate precursory state for meiotic recombination. Furthermore, the inactive X chromosome shows a progressive demethylation and reactivation, albeit partially. These findings establish the germline competence of hPSCs and provide a critical step toward human in vitro gametogenesis.〈/p〉
    Print ISSN: 0036-8075
    Electronic ISSN: 1095-9203
    Topics: Biology , Chemistry and Pharmacology , Computer Science , Medicine , Natural Sciences in General , Physics
    Published by American Association for the Advancement of Science (AAAS)
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  • 6
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    Generation of human oogonia from induced pluripotent stem cells in vitro (2018)
    American Association for the Advancement of Science (AAAS)
    In: Science
    Details
    Publication Date: 2018-10-19
    Description: Human in vitro gametogenesis may transform reproductive medicine. Human pluripotent stem cells (hPSCs) have been induced into primordial germ cell–like cells (hPGCLCs); however, further differentiation to a mature germ cell has not been achieved. Here, we show that hPGCLCs differentiate progressively into oogonia-like cells during a long-term in vitro culture (approximately 4 months) in xenogeneic reconstituted ovaries with mouse embryonic ovarian somatic cells. The hPGCLC-derived oogonia display hallmarks of epigenetic reprogramming—genome-wide DNA demethylation, imprint erasure, and extinguishment of aberrant DNA methylation in hPSCs—and acquire an immediate precursory state for meiotic recombination. Furthermore, the inactive X chromosome shows a progressive demethylation and reactivation, albeit partially. These findings establish the germline competence of hPSCs and provide a critical step toward human in vitro gametogenesis.
    Keywords: Development
    Print ISSN: 0036-8075
    Electronic ISSN: 1095-9203
    Topics: Biology , Chemistry and Pharmacology , Geosciences , Computer Science , Medicine , Natural Sciences in General , Physics
    Published by American Association for the Advancement of Science (AAAS)
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