EMBL Mouse Biology Unit

Monterotondo, Italy

EMBL Mouse Biology Unit

Monterotondo, Italy
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Kaar A.,University College Cork | Morley S.J.,University College Cork | Morley S.J.,EMBL Mouse Biology Unit | Rae M.G.,University College Cork
Journal of Neuroscience Methods | Year: 2017

Background Primary culture of postnatal central neurons is a widely used methodology for applications such as the investigation of neuronal development, protein trafficking/distribution and cellular signalling. However, successful production and maintenance of such cultures, particularly from postnatal animals, can be challenging. In attempting to surmount these difficulties, several disparate culturing methodologies have been developed. Such methodologies are centred on the identification and optimisation of critical steps and, as such, the protocols and reagents utilised can differ quite markedly from protocol to protocol, often with the suggestion that the use of a (usually expensive) proprietary reagent(s), lengthy substrate preparation and/or cell isolation techniques is/are necessary for successful culture preparation. New method Herein, we present a simple and inexpensive protocol for the preparation of primary hippocampal neurons from postnatal (2–5 day old) mice, which remain viable for experimental use for over one month. Results Neurons cultured using this method follow well established developmental norms and display typical responses to standard physiological stimuli such as depolarisation and certain pharmacological agents. Comparison with existing methods/conclusion By using a novel trituration technique, simplified methodology and non-proprietary reagents, we have developed a reliable protocol that enables the cost effective and efficient production of high quality postnatal mouse hippocampal cultures. This method, if required, can also be utilised to prepare neurons both from other regions of the brain as well as from other species such as rat. © 2017 Elsevier B.V.

Cornacchia D.,EMBL Mouse Biology Unit | Dileep V.,Florida State University | Quivy J.-P.,University Pierre and Marie Curie | Quivy J.-P.,French National Center for Scientific Research | And 8 more authors.
EMBO Journal | Year: 2012

The eukaryotic genome is replicated according to a specific spatio-temporal programme. However, little is known about both its molecular control and biological significance. Here, we identify mouse Rif1 as a key player in the regulation of DNA replication timing. We show that Rif1 deficiency in primary cells results in an unprecedented global alteration of the temporal order of replication. This effect takes place already in the first S-phase after Rif1 deletion and is neither accompanied by alterations in the transcriptional landscape nor by major changes in the biochemical identity of constitutive heterochromatin. In addition, Rif1 deficiency leads to both defective G1/S transition and chromatin re-organization after DNA replication. Together, these data offer a novel insight into the global regulation and biological significance of the replication-timing programme in mammalian cells. © 2012 European Molecular Biology Organization | All Rights Reserved.

Cerase A.,EMBL Mouse Biology Unit | Pintacuda G.,University of Oxford | Tattermusch A.,University of Oxford | Avner P.,EMBL Mouse Biology Unit | Avner P.,Institute Pasteur Paris
Genome Biology | Year: 2015

In female m ammals, one of the two X chromosomes in each cell is transcriptionally silenced in order to achieve dosage compensation between the genders in a process called X chromosome inactivation. The master regulator of this process is the long non-coding RNA Xist. During X-inactivation, Xist accumulates in cis on the future inactive X chromosome, triggering a cascade of events that provoke the stable silencing of the entire chromosome, with relatively few genes remaining active. How Xist spreads, what are its binding sites, how it recruits silencing factors and how it induces a specific topological and nuclear organization of the chromatin all remain largely unanswered questions. Recent studies have improved our understanding of Xist localization and the proteins with which it interacts, allowing a reappraisal of ideas about Xist function. We discuss recent advances in our knowledge of Xist-mediated silencing, focusing on Xist spreading, the nuclear organization of the inactive X chromosome, recruitment of the polycomb complex and the role of the nuclear matrix in the process of X chromosome inactivation. © 2015 Cerase et al.

Bereshchenko O.,EMBL Mouse Biology Unit | Bereshchenko O.,University of Perugia | Mancini E.,EMBL Mouse Biology Unit | Luciani L.,EMBL Mouse Biology Unit | And 5 more authors.
Haematologica | Year: 2012

Pontin is a highly conserved DNA helicase/ATPase which is a component of several macromolecular complexes with functions that include DNA repair, telomere maintenance and tumor suppression. While Pontin is known to be essential in yeast, fruit flies and frogs, its physiological role in mammalian organisms remains to be determined. We here find that Pontin is highly expressed in embryonic stem cells and hematopoietic tissues. Through germline inactivation of Ruvbl1, the gene encoding Pontin, we found it to be essential for early embryogenesis, as Ruvbl1 null embryos could not be recovered beyond the blastocyst stage where proliferation of the pluripotent inner cell mass was impaired. Conditional ablation of Ruvbl1 in hematopoietic tissues led to bone marrow failure. Competitive repopulation experiments showed that this included the loss of hematopoietic stem cells through apopotosis. Pontin is, therefore, essential for the function of both embryonic pluripotent cells and adult hematopoietic stem cells. ©2012 Ferrata Storti Foundation.

Becker S.,Robert Bosch GmbH | Becker S.,University of Tübingen | Oelschlaeger T.A.,University of Würzburg | Wullaert A.,University of Cologne | And 7 more authors.
PLoS ONE | Year: 2013

Background: The human colon harbours a plethora of bacteria known to broadly impact on mucosal metabolism and function and thought to be involved in inflammatory bowel disease pathogenesis and colon cancer development. In this report, we investigated the effect of colonic bacteria on epithelial cell differentiation factors in vitro and in vivo. As key transcription factors we focused on Hes1, known to direct towards an absorptive cell fate, Hath1 and KLF4, which govern goblet cell. Methods: Expression of the transcription factors Hes1, Hath1 and KLF4, the mucins Muc1 and Muc2 and the defensin HBD2 were measured by real-time PCR in LS174T cells following incubation with several heat-inactivated E. coli strains, including the probiotic E. coli Nissle 1917+/- flagellin, Lactobacilli and Bifidobacteria. For protein detection Western blot experiments and chamber-slide immunostaining were performed. Finally, mRNA and protein expression of these factors was evaluated in the colon of germfree vs. specific pathogen free vs. conventionalized mice and colonic goblet cells were counted. Results: Expression of Hes1 and Hath1, and to a minor degree also of KLF4, was reduced by E. coli K-12 and E. coli Nissle 1917. In contrast, Muc1 and HBD2 expression were significantly enhanced, independent of the Notch signalling pathway. Probiotic E. coli Nissle 1917 regulated Hes1, Hath1, Muc1 and HBD2 through flagellin. In vivo experiments confirmed the observed in vitro effects of bacteria by a diminished colonic expression of Hath1 and KLF4 in specific pathogen free and conventionalized mice as compared to germ free mice whereas the number of goblet cells was unchanged in these mice. Conclusions: Intestinal bacteria influence the intestinal epithelial differentiation factors Hes1, Hath1 and KLF4, as well as Muc1 and HBD2, in vitro and in vivo. The induction of Muc1 and HBD2 seems to be triggered directly by bacteria and not by Notch. © 2013 Becker et al.

Catela C.,EMBL Mouse Biology Unit | Catela C.,Mount Sinai School of Medicine | Kratsios P.,EMBL Mouse Biology Unit | Hede M.,EMBL Mouse Biology Unit | And 2 more authors.
Developmental Dynamics | Year: 2010

The unquestionable importance of the cardiovascular system for pre-and postnatal life has prompted dissection of the molecular mechanisms underlying its development. Serum and glucocorticoid-inducible kinase 1 (SGK1) is a serine/threonine kinase lying downstream of the phosphoinositide 3 (PI3) kinase pathway, whose embryonic function remains unknown. Here, we show that disruption of Sgk1 in the mouse C57BL/6J genetic background leads to embryonic lethality at embryonic day 10.5-11.5 due to severe embryonic and extraembryonic angiogenic defects and to impaired myocardial trabeculation. Absence of SGK1 results in increased apoptosis of endothelial cells, and of vascular smooth muscle cells highlighting a prosurvival role for SGK1 during angiogenesis. Sgk1 null embryos also display reduced expression levels of Notch signaling genes and decreased expression of the arterial markers Efnb2 and Nrp1. These findings uncover a novel and essential function for SGK1 in cardiovascular development contributing to a better understanding of mammalian angiogenesis. © 2010 Wiley-Liss, Inc.

Reckzeh K.,Lund University | Bereshchenko O.,EMBL Mouse Biology Unit | Mead A.,University of Oxford | Rehn M.,Lund University | And 5 more authors.
Leukemia | Year: 2012

Biallelic CEBPA mutations and FMS-like tyrosine kinase receptor 3 (FLT3) length mutations are frequently identified in human acute myeloid leukemia (AML) with normal cytogenetics. However, the molecular and cellular mechanisms of oncogene cooperation remain unclear because of a lack of disease models. We have generated an AML mouse model using knockin mouse strains to study cooperation of an internal tandem duplication (ITD) mutation in the Flt3 gene with commonly observed CCAAT/enhancer binding protein alpha (C/EBPα) mutations. This study provides evidence that FLT3 ITD cooperates in leukemogenesis by enhancing the generation of leukemia-initiating granulocyte-monocyte progenitors (GMPs) otherwise prevented by a block in differentiation and skewed lineage priming induced by biallelic C/EBPα mutations. These cellular changes are accompanied by an upregulation of hematopoietic stem cell and STAT5 target genes. By gene expression analysis in premalignant populations, we further show a role of FLT3 ITD in activating genes involved in survival/transformation and chemoresistance. Both multipotent progenitors and GMP cells contain the potential to induce AML similar to corresponding cells in human AML samples showing that this model resembles human disease. © 2012 Macmillan Publishers Limited.

Pintacuda G.,University of Oxford | Cerase A.,EMBL Mouse Biology Unit
Stem Cell Reviews and Reports | Year: 2015

X chromosome inactivation (XCI) is the dosage compensation mechanism that evolved in female mammals to correct the genetic imbalance of X-linked genes between sexes. X chromosome inactivation occurs in early development when one of the two X chromosomes of females is nearly-completely silenced. Differentiating Embryonic Stem cells (ESC) are regarded as a useful tool to study XCI, since they recapitulate many events occurring during early development. In this review we aim to summarise the advances in the field and to discuss the close connection between cell differentiation and X chromosome inactivation, with a particular focus on mouse ESCs. © 2015, The Author(s).

Mancini E.,EMBL Mouse Biology Unit | Sanjuan-Pla A.,University of Edinburgh | Luciani L.,EMBL Mouse Biology Unit | Moore S.,University of Edinburgh | And 9 more authors.
EMBO Journal | Year: 2012

The transcription factors that control lineage specification of haematopoietic stem cells (HSCs) have been well described for the myeloid and lymphoid lineages, whereas transcriptional control of erythroid (E) and megakaryocytic (Mk) fate is less understood. We here use conditional removal of the GATA-1 and FOG-1 transcription factors to identify FOG-1 as required for the formation of all committed Mk-and E-lineage progenitors, whereas GATA-1 was observed to be specifically required for E-lineage commitment. FOG-1-deficient HSCs and preMegEs, the latter normally bipotent for the Mk and E lineages, underwent myeloid transcriptional reprogramming, and formed myeloid, but not erythroid and megakaryocytic cells in vitro. These results identify FOG-1 and GATA-1 as required for formation of bipotent Mk/E progenitors and their E-lineage commitment, respectively, and show that FOG-1 mediates transcriptional Mk/E programming of HSCs as well as their subsequent Mk/E-lineage commitment. Finally, C/EBPs and FOG-1 exhibited transcriptional cross-regulation in early myelo-erythroid progenitors making their functional antagonism a potential mechanism for separation of the myeloid and Mk/E lineages. © 2012 European Molecular Biology Organization | All Rights Reserved.

S.B.C. B.,EMBL Mouse Biology Unit
Experimental Cell Research | Year: 2010

Constitutive heterochromatin is essential for chromosome maintenance in all eukaryotes. However, the repetitive nature of the underlying DNA, the presence of very stable protein-DNA complexes and the highly compacted nature of this type of chromatin represent a challenge for the DNA replication machinery. Data collected from different model organisms suggest that at least some of the components of the DNA replication checkpoint could be essential for ensuring the completion of DNA replication in the context of heterochromatin. I review and discuss the literature that directly or indirectly contributes to the formulation of this hypothesis. In particular, I focus my attention on Rif1, a newly discovered member of the DNA replication checkpoint. Recent data generated in mammalian cells highlight the spatial and temporal relation between Rif1, pericentromeric heterochromatin and S-phase. I review these recent and the previous data coming from studies performed in yeast in order to highlight the possible evolutionary conserved links and propose a molecular model for Rif1 role in heterochromatin replication. © 2010 Elsevier Inc.

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