scholarly journals BLISS: quantitative and versatile genome-wide profiling of DNA breaks in situ

2016 ◽  
Author(s):  
Winston X. Yan ◽  
Reza Mirzazadeh ◽  
Silvano Garnerone ◽  
David Scott ◽  
Martin W. Schneider ◽  
...  
Keyword(s):  
High Efficiency ◽  
Embryonic Stem ◽  
Dna Breaks ◽  
Drug Induced ◽  
Low Input ◽  
Tissue Sections ◽  
Genome Wide ◽  
Primary Mouse

AbstractWe present a method for genome-wide DNA double-strand Breaks (DSBs) Labeling In Situ and Sequencing (BLISS) which, compared to existing methods, introduces several key features: 1) high efficiency and low input requirement by in situ DSB labeling in cells or tissue sections directly on a solid surface; 2) easy scalability by performing in situ reactions in multi-well plates; 3) high sensitivity by linearly amplifying tagged DSBs using in vitro transcription; and 4) accurate DSB quantification and control of PCR biases by using unique molecular identifiers. We demonstrate the ability to use BLISS to quantify natural and drug-induced DSBs in low-input samples of cancer cells, primary mouse embryonic stem cells, and mouse liver tissue sections. Finally, we applied BLISS to compare the specificity of CRISPR-associated RNA-guided endonucleases Cas9 and Cpf1, and found that Cpf1 has higher specificity than Cas9. These results establish BLISS as a versatile, sensitive, and efficient method for genome-wide DSB mapping in many applications.

scholarly journals Breaks Labeling in situ and sequencing (BLISS)

2019 ◽  
Author(s):  
Winston X. Yan ◽  
Reza Mirzazadeh ◽  
Silvano Garnerone ◽  
David Scott ◽  
Martin W. Schneider ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Low Input ◽  
Tissue Sections ◽  
Genome Wide ◽  
Transcription 3 ◽  
Target Activity

Abstract Precisely measuring the location and frequency of DNA double-strand breaks \(DSBs) along the genome is instrumental to understanding genomic fragility, but current methods are limited in versatility, sensitivity, or practicality. Here, we present Breaks Labeling _In Situ_ and Sequencing \(BLISS), featuring: 1) direct labeling of DSBs in fixed cells or tissue sections on a solid surface; 2) low-input requirement by linear amplification of tagged DSBs by _in vitro_ transcription; 3) quantification of DSBs through unique molecular identifiers; and 4) easy scalability and multiplexing. We apply BLISS to profile endogenous and exogenous DSBs in low-input samples of cancer cells, embryonic stem cells, and liver tissue. We demonstrate the sensitivity of BLISS by assessing the genome-wide off-target activity of two CRISPR-associated RNA-guided endonucleases, Cas9 and Cpf1, observing that Cpf1 has higher specificity than Cas9. Our results establish BLISS as a versatile, sensitive, and efficient method for genome-wide DSB mapping in many applications. W.Yan and R.Mirzazadeh equally contributed to this work.

scholarly journals Induction of Recurrent Break Cluster Genes in Neural Progenitor Cells Differentiated from Embryonic Stem Cells In Culture

2019 ◽  
Author(s):  
Aseda Tena ◽  
Yuxiang Zhang ◽  
Nia Kyritsis ◽  
Anne Devorak ◽  
Jeffrey Zurita ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Progenitor Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Neural Progenitors ◽  
Neural Genes ◽  
Genome Wide ◽  
Primary Mouse

ABSTRACTMild replication stress enhances appearance of dozens of robust recurrent genomic break clusters, termed RDCs, in cultured primary mouse neural stem and progenitor cells (NSPCs). Robust RDCs occur within genes (“RDC-genes”) that are long and have roles in neural cell communications and/or have been implicated in neuropsychiatric diseases or cancer. We sought to develop an in vitro approach to determine whether specific RDC formation is associated with neural development. For this purpose, we adapted a system to induce neural progenitor cell (NPC) development from mouse embryonic stem cell (ESC) lines deficient for XRCC4 plus p53, a genotype that enhances DNA double-strand break (DSB) persistence to enhance detection. We tested for RDCs by our genome wide DSB identification approach that captures DSBs genome-wide via their ability to join to specific genomic Cas9/sgRNA-generated bait DSBs. In XRCC4/p53-deficient ES cells, we detected 7 RDCs, which were in genes, with two RDCs being robust. In contrast, in NPCs derived from these ES cell lines, we detected 29 RDCs, a large fraction of which were robust and associated with long, transcribed neural genes that were also robust RDC-genes in primary NSPCs. These studies suggest that many RDCs present in NSPCs are developmentally influenced to occur in this cell type and indicate that induced development of NPCs from ES cells provides an approach to rapidly elucidate mechanistic aspects of NPC RDC formation.SIGNIFICANCE STATEMENTWe previously discovered a set of long neural genes susceptible to frequent DNA breaks in primary mouse brain progenitor cells. We termed these genes RDC-genes. RDC-gene breakage during brain development might alter neural gene function and contribute to neurological diseases and brain cancer. To provide an approach to characterize the unknown mechanism of neural RDC-gene breakage, we asked whether RDC-genes appear in neural progenitors differentiated from embryonic stem cells in culture. Indeed, robust RDC-genes appeared in neural progenitors differentiated in culture and many overlapped with robust RDC-genes in primary brain progenitors. These studies indicate that in vitro development of neural progenitors provides a model system for elucidating how RDC-genes are formed.

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 Genome-Wide Binding Analyses of HOXB1 Revealed a Novel DNA Binding Motif Associated with Gene Repression

2021 ◽  
Vol 9 (1) ◽  
pp. 6
Author(s):  
Narendra Pratap Singh ◽  
Bony De Kumar ◽  
Ariel Paulson ◽  
Mark E. Parrish ◽  
Carrie Scott ◽  
...  
Keyword(s):  
Dna Binding ◽  
Target Genes ◽  
Embryonic Stem ◽  
Binding Motif ◽  
Binding Assays ◽  
Histone Marks ◽  
Genome Wide ◽  
Hox Cofactors

Knowledge of the diverse DNA binding specificities of transcription factors is important for understanding their specific regulatory functions in animal development and evolution. We have examined the genome-wide binding properties of the mouse HOXB1 protein in embryonic stem cells differentiated into neural fates. Unexpectedly, only a small number of HOXB1 bound regions (7%) correlate with binding of the known HOX cofactors PBX and MEIS. In contrast, 22% of the HOXB1 binding peaks display co-occupancy with the transcriptional repressor REST. Analyses revealed that co-binding of HOXB1 with PBX correlates with active histone marks and high levels of expression, while co-occupancy with REST correlates with repressive histone marks and repression of the target genes. Analysis of HOXB1 bound regions uncovered enrichment of a novel 15 base pair HOXB1 binding motif HB1RE (HOXB1 response element). In vitro template binding assays showed that HOXB1, PBX1, and MEIS can bind to this motif. In vivo, this motif is sufficient for direct expression of a reporter gene and over-expression of HOXB1 selectively represses this activity. Our analyses suggest that HOXB1 has evolved an association with REST in gene regulation and the novel HB1RE motif contributes to HOXB1 function in part through a repressive role in gene expression.

In vitro assessing the risk of drug-induced cardiotoxicity by embryonic stem cell-based biosensor

2011 ◽  
Vol 155 (1) ◽  
pp. 214-219 ◽  
Author(s):  
Qingjun Liu ◽  
Hui Yu ◽  
Zhou Tan ◽  
Hua Cai ◽  
Weiwei Ye ◽  
...  
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Embryonic Stem ◽  
Drug Induced

scholarly journals A new method to detect apoptosis in paraffin sections: in situ end-labeling of fragmented DNA.

1993 ◽  
Vol 41 (1) ◽  
pp. 7-12 ◽  
Author(s):  
J H Wijsman ◽  
R R Jonker ◽  
R Keijzer ◽  
C J van de Velde ◽  
C J Cornelisse ◽  
...  
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Staining Method ◽  
Morphological Characteristics ◽  
Image Cytometry ◽  
Apoptotic Cells ◽  
Dna Breaks ◽  
Paraffin Sections ◽  
Tissue Sections

Apoptosis (programmed cell death) can be difficult to detect in routine histological sections. Since extensive DNA fragmentation is an important characteristic of this process, visualization of DNA breaks could greatly facilitate the identification of apoptotic cells. We describe a new staining method for formalin-fixed, paraffin-embedded tissue sections that involves an in situ end-labeling (ISEL) procedure. After protease treatment to permeate the tissue sections, biotinylated nucleotides are in situ incorporated into DNA breaks by polymerase and subsequently stained with DAB via peroxidase-conjugated avidin. Staining of cells with the morphological characteristics of apoptosis was demonstrated in tissues known to exhibit programmed cell death, i.e., prostate and uterus after castration, tumors, lymph node follicles, and embryos. Apoptotic cells could be discriminated morphologically from areas of labeled necrotic cells, in which DNA degradation also occurs. Because apoptosis is relatively easily recognized in H&E-stained sections of involuting prostates of castrated rats, we used this model system to validate the ISEL method for the quantification of apoptotic cells. A high correlation was found between the fractions of ISEL-labeled cells and the fractions of apoptotic cells that were morphologically determined in adjacent sections. We conclude that ISEL is a useful technique for quantification of apoptosis in paraffin sections, especially for those tissues in which morphological determination is difficult. Furthermore, this new staining method enables the use of automated image cytometry for evaluating apoptosis.

scholarly journals Targeted in situ genome-wide profiling with high efficiency for low cell numbers

2018 ◽  
Vol 13 (5) ◽  
pp. 1006-1019 ◽  
Author(s):  
Peter J Skene ◽  
Jorja G Henikoff ◽  
Steven Henikoff
Keyword(s):  
High Efficiency ◽  
Cell Numbers ◽  
Genome Wide

Novel Materials for Myco-Decontamination of Cyanide-Containing Wastewaters through Microbial Biotechnology

2021 ◽  
Vol 1037 ◽  
pp. 751-758
Author(s):  
Igor N. Pavlov ◽  
Yulia A. Litovka
Keyword(s):  
Large Scale ◽  
High Efficiency ◽  
Pine Sawdust ◽  
Microbial Biotechnology ◽  
Tailing Dam ◽  
Large Scale Cultivation ◽  
Siberian Pine ◽  
Uniform Ratio

This study examined the effectiveness of decontamination of industrial cyanide-containing water using mycelium-based lignocellulosic materials. These results suggest that fungi biomass and plant substrates can be used successfully in the treatment of wastewater contaminated by cyanide. Fungi were isolated from old wood samples taken from a tailing dam with high cyanide content (more than 20 years in semi-submerged condition). All isolated fungi belonged to the genus Fusarium. Fusarium oxysporum Schltdl. is most effective for biodegradation of cyanide-containing wastewaters (even at low temperatures). The most optimal lignocellulosic composition for production of mycelium-based biomaterial for biodegradation of cyanide wastewater consists of a uniform ratio of Siberian pine sawdust and wheat straw. The high efficiency of mycelium-based materials has been experimentally proven in vitro at 15-25 ° C. New fungal biomaterials are provide decrease in the concentration of cyanide ions to 79% (P <0.001). Large-scale cultivation of fungi biomass was carried out by the periodic liquid-phase cultivation. The submerged biomass from bioreactor was used as an inoculum for the production of mycelium-based materials for bioremediation of cyanide wastewater in situ (gold mine tailing).

scholarly journals Cell stemness is maintained upon concurrent expression of RB and the mitochondrial ribosomal protein S18-2

2020 ◽  
Vol 117 (27) ◽  
pp. 15673-15683
Author(s):  
Muhammad Mushtaq ◽  
Larysa Kovalevska ◽  
Suhas Darekar ◽  
Alexandra Abramsson ◽  
Henrik Zetterberg ◽  
...  
Keyword(s):  
Stem Cell ◽  
Ribosomal Protein ◽  
Severe Combined Immunodeficiency ◽  
Ectopic Expression ◽  
Embryonic Stem ◽  
Ring Finger ◽  
Histone H2a ◽  
Mitochondrial Ribosomal Protein ◽  
Primary Mouse

Stemness encompasses the capability of a cell for self-renewal and differentiation. The stem cell maintains a balance between proliferation, quiescence, and regeneration via interactions with the microenvironment. Previously, we showed that ectopic expression of the mitochondrial ribosomal protein S18-2 (MRPS18-2) led to immortalization of primary fibroblasts, accompanied by induction of an embryonic stem cell (ESC) phenotype. Moreover, we demonstrated interaction between S18-2 and the retinoblastoma-associated protein (RB) and hypothesized that the simultaneous expression of RB and S18-2 is essential for maintaining cell stemness. Here, we experimentally investigated the role of S18-2 in cell stemness and differentiation. Concurrent expression of RB and S18-2 resulted in immortalization ofRb1−/−primary mouse embryonic fibroblasts and in aggressive tumor growth in severe combined immunodeficiency mice. These cells, which express both RB and S18-2 at high levels, exhibited the potential to differentiate into various lineages in vitro, including osteogenic, chondrogenic, and adipogenic lineages. Mechanistically, S18-2 formed a multimeric protein complex with prohibitin and the ring finger protein 2 (RNF2). This molecular complex increased the monoubiquitination of histone H2ALys119, a characteristic trait of ESCs, by enhanced E3-ligase activity of RNF2. Furthermore, we found enrichment of KLF4 at theS18-2promoter region and that theS18-2expression is positively correlated withKLF4levels. Importantly, knockdown of S18-2 in zebrafish larvae led to embryonic lethality. Collectively, our findings suggest an important role for S18-2 in cell stemness and differentiation and potentially also in cancerogenesis.

scholarly journals The Dysregulated Galectin Network Activates NF-κB to Induce Disease Markers and Matrix Degeneration in 3D Pellet Cultures of Osteoarthritic Chondrocytes

2020 ◽  
Author(s):  
K. M. Pichler ◽  
D. Weinmann ◽  
S. Schmidt ◽  
B. Kubista ◽  
R. Lass ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Disease Model ◽  
Caffeic Acid Phenethyl Ester ◽  
Pellet Size ◽  
Tissue Sections ◽  
Osteoarthritic Chondrocytes ◽  
2D And 3D

AbstractThis work aimed to study the dysregulated network of galectins in OA chondrocyte pellets, and to assess whether their recently discovered activity as molecular switches of functional biomarkers results in degradation of extracellular matrix in vitro. Scaffold-free 3D pellet cultures were established of human OA chondrocytes. Expression and secretion of galectin(Gal)-1, -3, and -8 were monitored relative to 2D cultures or clinical tissue sections by RT-qPCR, immunohistochemistry and ELISAs. Exposure of 2D and 3D cultures to an in vivo-like galectin mixture (Gal-1 and Gal-8: 5 µg/ml, Gal-3: 1 µg/ml) was followed by the assessment of pellet size, immunohistochemical matrix staining, and/or quantification of MMP-1, -3, and -13. Application of inhibitors of NF-κB activation probed into the potential of intervening with galectin-induced matrix degradation. Galectin profiling revealed maintained dysregulation of Gal-1, -3, and -8 in pellet cultures, resembling the OA situation in situ. The presence of the galectin mixture promoted marked reduction of pellet size and loss of collagen type II-rich extracellular matrix, accompanied by the upregulation of MMP-1, -3, and -13. Inhibition of p65-phosphorylation by caffeic acid phenethyl ester effectively alleviated the detrimental effects of galectins, resulting in downregulated MMP secretion, reduced matrix breakdown and augmented pellet size. This study suggests that the dysregulated galectin network in OA cartilage leads to extracellular matrix breakdown, and provides encouraging evidence of the feasible inhibition of galectin-triggered activities. OA chondrocyte pellets have the potential to serve as in vitro disease model for further studies on galectins in OA onset and progression.

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