scholarly journals A Targeted Deletion of a Region Upstream from the Jκ Cluster Impairs κ Chain Rearrangement In Cis in Mice and in the 103/bcl2 Cell Line

1999 ◽  
Vol 189 (9) ◽  
pp. 1443-1450 ◽  
Author(s):  
Laurentiu Cocea ◽  
Annie De Smet ◽  
Mahasti Saghatchian ◽  
Simon Fillatreau ◽  
Laurent Ferradini ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Cell Line ◽  
Embryonic Stem ◽  
Similar Reduction ◽  
Targeted Deletion ◽  
Vitro Model ◽  
Germline Transcript ◽  
In Cis

We have shown previously that a mutation of the KI-KII site immediately 5′ to Jκ1 on the mouse immunoglobulin light chain κ locus reduces the rearrangement level in cis, although it does not affect transcription. Here we deleted by homologous recombination in mouse embryonic stem cells a 4-kb DNA fragment, located immediately upstream of the KI-KII element, which contains the promoter of the long germline transcript. Analysis of gene-targeted heterozygous mouse splenic B cells showed a strong decrease in rearrangement for the allele bearing the deletion. When both the KI-KII mutation and the 4-kb deletion were present on the same allele, the overall reduction in rearrangement was stronger than with the 4-kb deletion alone underlying the role of these two elements in the regulation of rearrangement. The same deletion was performed by homologous recombination on one allele of the rearrangement- inducible mouse 103/bcl2-hygroR pre-B cell line, and resulted in a similar reduction in the induction of rearrangement of the mutated allele. This result validates this cell line as an in vitro model for studying the incidence of gene-targeted modifications of the κ locus on the regulation of rearrangement.

scholarly journals EOMES is responsible for WNT memory and can substitute for WNT in mesendoderm specification

2021 ◽  
Author(s):  
Anna Yoney ◽  
Lu Bai ◽  
Ali H. Brivanlou ◽  
Eric D Siggia
Keyword(s):  
Target Genes ◽  
Embryonic Stem ◽  
Model Systems ◽  
Mechanistic Basis ◽  
Vitro Model ◽  
Transcriptional Output ◽  
Accessible Chromatin ◽  
Pluripotency Maintenance

Embryogenesis is guided by a limited set of signaling pathways that are reused at different times and places throughout development. How a context dependent signaling response is generated has been a central question of developmental biology, which can now be addressed with in vitro model systems. Our previous work in human embryonic stem cells (hESCs) established that pre-exposure of cells to WNT/β-catenin signaling is sufficient to switch the output of ACTIVIN/SMAD2 signaling from pluripotency maintenance to mesendoderm (ME) differentiation. A body of previous literature has established the role of both pathways in ME differentiation. However, our work demonstrated that the two signals do not need to be present simultaneously and that hESCs have a means to record WNT signals. Here we demonstrate that hESCs have accessible chromatin at SMAD2 binding sites near pluripotency and ME-associated target genes and that WNT priming does not alter SMAD2 binding. Rather our results indicate that stable transcriptional output at ME genes results from WNT-dependent production of an additional SMAD2 co-factor, EOMES. We show that expression of EOMES can replace WNT signaling in ME differentiation, providing a mechanistic basis for WNT-priming and memory in early development.

The Role of Livin, An Inhibitor of Apoptosis Protein, in Thrombopoiesis.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1842-1842
Author(s):  
Ihab Abd-Elrahman ◽  
Varda Deutsch ◽  
Klilah Hershko ◽  
Tsahi Noyman ◽  
Itay Lazar ◽  
...  
Keyword(s):  
Cell Line ◽  
Apoptotic Cell ◽  
Erythroid Cell ◽  
Inhibitor Of Apoptosis ◽  
Inhibitor Of Apoptosis Protein ◽  
Platelet Production ◽  
Apoptosis Protein ◽  
Vitro Model

Abstract The exact mechanism of platelet production is poorly understood. A relationship between activation of the apoptotic cell machinery and the formation of pro-platelets has been established. In this study we show that Livin plays a major role in this process. Livin is a member of the Inhibitor of Apoptosis Protein (IAP) family, a novel family of intracellular anti-apoptotic proteins that act by binding and inhibiting caspases. We found that Livin mRNA and protein are expressed in platelets and Livin was also clearly detected by immunohistochemistry staining in normal mature bone marrow megakaryocytes (MK). Overexpression of Livin was also demonstrated in MK of patients with various hematological diseases such as ITP, MDS, Hodgkin’s disease, ET and PV. An in vitro model was established to evaluate the potential role of Livin in thrombopoiesis. The human erythroleukemic cell line, LAMA-84, was induced by a phorbol ester (PMA) to differentiate to MK. Differentiation was characterized by upregulation of the MK marker CD41 from 6% to >60% and downregulation of the erythroid cell marker CD71 from 97% to 70%. Increased cell size, ploidy, and DNA synthesis, all markers of MK differentiation, were detected by flow cytometry. Upon differentiation induced by PMA, LAMA-84 cells formed pro-platelets and produced functional platelets capable of aggregation. This thrombopoiesis was accompanied by Livin overexpression. Moreover, overexpression of Livin by transfection decreased the percentage of the undifferentiated CD71+ cells to 53 % compared to 73 % in control cells (p= 0.007) and increased the production of platelets by two folds compared to control cells. Overexpression of Livin reduced caspase 3 activity and inhibited MK apoptosis. Our results show that anti-apoptotic Livin plays a role in thrombopoiesis possibly by protecting the maturing MK from apoptosis, thus affording more time for efficient platelet production by longer living pro-platelets. We are currently using this new cell line model to further characterize the molecular relationship between apoptosis and thrombopoiesis.

Targeted deletion of the CD59 gene causes spontaneous intravascular hemolysis and hemoglobinuria

Blood ◽  
2001 ◽  
Vol 98 (2) ◽  
pp. 442-449 ◽  
Author(s):  
Dewi S. Holt ◽  
Marina Botto ◽  
Anne E. Bygrave ◽  
S. Melanie Hanna ◽  
Mark J. Walport ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Membrane Attack Complex ◽  
Intravascular Hemolysis ◽  
Targeted Deletion ◽  
Circulating Cells ◽  
Health And Disease ◽  
Fresh Plasma ◽  
Host Tissues

The glycolipid-anchored glycoprotein CD59 inhibits assembly of the lytic membrane attack complex of complement by incorporation into the forming complex. Absence of CD59 and other glycolipid-anchored molecules on circulating cells in the human hemolytic disorder paroxysmal nocturnal hemoglobinuria is associated with intravascular hemolysis and thrombosis. To examine the role of CD59 in protecting host tissues in health and disease, CD59-deficient (CD59−/−) mice were produced by gene targeting in embryonic stem cells. Absence of CD59 was confirmed by staining cells and tissues with specific antibody. Despite the complete absence of CD59, mice were healthy and fertile. Erythrocytes in vitro displayed increased susceptibility to complement and were positive in an acidified serum lysis test. Despite this, CD59−/− mice were not anemic but had elevated reticulocyte counts, indicating accelerated erythrocyte turnover. Fresh plasma and urine from CD59−/− mice contained increased amounts of hemoglobin when compared with littermate controls, providing further evidence for spontaneous intravascular hemolysis. Intravascular hemolysis was increased following administration of cobra venom factor to trigger complement activation. CD59−/− mice will provide a tool for characterizing the importance of CD59 in protection of self tissues from membrane attack complex damage in health and during diseases in which complement is activated.

scholarly journals Construction of a mammalian embryo model from stem cells organized by a morphogen signalling centre

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Peng-Fei Xu ◽  
Ricardo Moraes Borges ◽  
Jonathan Fillatre ◽  
Maraysa de Oliveira-Melo ◽  
Tao Cheng ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Tracking ◽  
Cardiac Tissue ◽  
Embryonic Stem ◽  
Mammalian Embryo ◽  
Wide Range ◽  
Vitro Model ◽  
Neurula Stage

AbstractGenerating properly differentiated embryonic structures in vitro from pluripotent stem cells remains a challenge. Here we show that instruction of aggregates of mouse embryonic stem cells with an experimentally engineered morphogen signalling centre, that functions as an organizer, results in the development of embryo-like entities (embryoids). In situ hybridization, immunolabelling, cell tracking and transcriptomic analyses show that these embryoids form the three germ layers through a gastrulation process and that they exhibit a wide range of developmental structures, highly similar to neurula-stage mouse embryos. Embryoids are organized around an axial chordamesoderm, with a dorsal neural plate that displays histological properties similar to the murine embryo neuroepithelium and that folds into a neural tube patterned antero-posteriorly from the posterior midbrain to the tip of the tail. Lateral to the chordamesoderm, embryoids display somitic and intermediate mesoderm, with beating cardiac tissue anteriorly and formation of a vasculature network. Ventrally, embryoids differentiate a primitive gut tube, which is patterned both antero-posteriorly and dorso-ventrally. Altogether, embryoids provide an in vitro model of mammalian embryo that displays extensive development of germ layer derivatives and that promises to be a powerful tool for in vitro studies and disease modelling.

scholarly journals Antagonism of Adherent Invasive E. coli LF82 With Human α-defensin 5 in the Follicle-associated Epithelium of Patients With Ileal Crohn’s Disease

2020 ◽  
Author(s):  
Lina Y Alkaissi ◽  
Martin E Winberg ◽  
Stéphanie DS Heil ◽  
Staffan Haapaniemi ◽  
Pär Myrelid ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Ex Vivo ◽  
Ussing Chambers ◽  
E Coli ◽  
Follicle Associated Epithelium ◽  
Vitro Model ◽  
Set Up

Abstract Background The first visible signs of Crohn’s disease (CD) are microscopic erosions over the follicle-associated epithelium (FAE). The aim of the study was to investigate the effects of human α-defensin 5 (HD5) on adherent-invasive Escherichia coli LF82 translocation and HD5 secretion after LF82 exposure in an in vitro model of human FAE and in human FAE ex vivo. Methods An in vitro FAE-model was set up by the coculture of Raji B cells and Caco-2-cl1 cells. Ileal FAE from patients with CD and controls were mounted in Ussing chambers. The effect of HD5 on LF82 translocation was studied by LF82 exposure to the cells or tissues with or without incubation with HD5. The HD5 secretion was measured in human FAE exposed to LF82 or Salmonella typhimurium. The HD5 levels were evaluated by immunofluorescence, immunoblotting, and ELISA. Results There was an increased LF82 translocation across the FAE-model compared with Caco-2-cl1 (P < 0.05). Incubation of cell/tissues with HD5 before LF82 exposure reduced bacterial passage in both models. Human FAE showed increased LF82 translocation in CD compared with controls and attenuated passage after incubation with sublethal HD5 in both CD and controls (P < 0.05). LF82 exposure resulted in a lower HD5 secretion in CD FAE compared with controls (P < 0.05), whereas Salmonella exposure caused equal secretion on CD and controls. There were significantly lower HD5 levels in CD tissues compared with controls. Conclusions Sublethal HD5 reduces the ability of LF82 to translocate through FAE. The HD5 is secreted less in CD in response to LF82, despite a normal response to Salmonella. This further implicates the integrated role of antimicrobial factors and barrier function in CD pathogenesis.

scholarly journals Oxygen tension modulates the mitochondrial genetic bottleneck and influences the segregation of a heteroplasmic mtDNA variant in vitro

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Mikael G. Pezet ◽  
Aurora Gomez-Duran ◽  
Florian Klimm ◽  
Juvid Aryaman ◽  
Stephen Burr ◽  
...  
Keyword(s):  
Germ Cell ◽  
Oxygen Tension ◽  
Germ Line ◽  
Embryonic Stem ◽  
Mitochondrial Diseases ◽  
Genetic Bottleneck ◽  
Cellular Mechanisms ◽  
Germ Cell Specification ◽  
Vitro Model

AbstractMost humans carry a mixed population of mitochondrial DNA (mtDNA heteroplasmy) affecting ~1–2% of molecules, but rapid percentage shifts occur over one generation leading to severe mitochondrial diseases. A decrease in the amount of mtDNA within the developing female germ line appears to play a role, but other sub-cellular mechanisms have been implicated. Establishing an in vitro model of early mammalian germ cell development from embryonic stem cells, here we show that the reduction of mtDNA content is modulated by oxygen and reaches a nadir immediately before germ cell specification. The observed genetic bottleneck was accompanied by a decrease in mtDNA replicating foci and the segregation of heteroplasmy, which were both abolished at higher oxygen levels. Thus, differences in oxygen tension occurring during early development likely modulate the amount of mtDNA, facilitating mtDNA segregation and contributing to tissue-specific mutation loads.

The Human Leukemic HL-60 Cell Line: An in Vitro Model for Cell Death Endpoint Identification

1996 ◽  
Vol 24 (4) ◽  
pp. 581-587
Author(s):  
Cristiana Zanetti ◽  
Arrnalaura Stammati ◽  
Orazio Sapora ◽  
Flavia Zucco
Keyword(s):  
Cell Death ◽  
Cell Line ◽  
In Vitro Model ◽  
Leukemia Cell ◽  
Promyelocytic Leukemia ◽  
Leukemia Cell Line ◽  
Cell Type ◽  
Vitro Model ◽  
Promyelocytic Leukemia Cell Line

The aim of this study was to investigate the endpoints related to cell death, either necrosis or apoptosis, induced by four chemicals in the promyelocytic leukemia cell line, HL-60. Cell morphology, DNA fragmentation, cytofluorimetric analysis and oxygen consumption were used to classify the type of cell death observed. In our analysis, we found that not all the selected parameters reproduced the differences observed in the cell death caused by the four chemicals tested. As cell death is a very complex phenomenon, several factors should be taken into account (cell type, exposure time and chemical concentration), if chemicals are to be classified according to differences in the mechanisms more directly involved in cell death.

scholarly journals Role of the C5a-C5a receptor axis in the inflammatory responses of the lungs after experimental polytrauma and hemorrhagic shock

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shinjini Chakraborty ◽  
Veronika Eva Winkelmann ◽  
Sonja Braumüller ◽  
Annette Palmer ◽  
Anke Schultze ◽  
...  
Keyword(s):  
Hemorrhagic Shock ◽  
Inflammatory Responses ◽  
Experimental Models ◽  
Lung Damage ◽  
C5a Receptor ◽  
Cytokine Levels ◽  
Vitro Model

AbstractSingular blockade of C5a in experimental models of sepsis is known to confer protection by rescuing lethality and decreasing pro-inflammatory responses. However, the role of inhibiting C5a has not been evaluated in the context of sterile systemic inflammatory responses, like polytrauma and hemorrhagic shock (PT + HS). In our presented study, a novel and highly specific C5a L-aptamer, NoxD21, was used to block C5a activity in an experimental murine model of PT + HS. The aim of the study was to assess early modulation of inflammatory responses and lung damage 4 h after PT + HS induction. NoxD21-treated PT + HS mice displayed greater polymorphonuclear cell recruitment in the lung, increased pro-inflammatory cytokine levels in the bronchoalveolar lavage fluids (BALF) and reduced myeloperoxidase levels within the lung tissue. An in vitro model of the alveolar-capillary barrier was established to confirm these in vivo observations. Treatment with a polytrauma cocktail induced barrier damage only after 16 h, and NoxD21 treatment in vitro did not rescue this effect. Furthermore, to test the exact role of both the cognate receptors of C5a (C5aR1 and C5aR2), experimental PT + HS was induced in C5aR1 knockout (C5aR1 KO) and C5aR2 KO mice. Following 4 h of PT + HS, C5aR2 KO mice had significantly reduced IL-6 and IL-17 levels in the BALF without significant lung damage, and both, C5aR1 KO and C5aR2 KO PT + HS animals displayed reduced MPO levels within the lungs. In conclusion, the C5aR2 could be a putative driver of early local inflammatory responses in the lung after PT + HS.

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