scholarly journals CAS-LiveFISH: Simple and versatile imaging of genomic loci in live mammalian cells and early pre-implantation embryos

2020 ◽  
Author(s):  
Yongtao Geng ◽  
Alexandros Pertsinidis
Keyword(s):  
Mammalian Cells ◽  
Target Gene ◽  
Nuclear Architecture ◽  
Live Cells ◽  
Transcription Activator ◽  
Distal Enhancer ◽  
Chromatin Dynamics ◽  
Guide Rnas ◽  
Early Mouse

SUMMARYVisualizing the 4D genome in live cells is essential for understanding its regulation. Programmable DNA-binding probes, such as fluorescent clustered regularly interspaced short palindromic repeats (CRISPR) and transcription activator-like effector (TALE) proteins have recently emerged as powerful tools for imaging specific genomic loci in live cells. However, many such systems rely on genetically-encoded components, often requiring multiple constructs that each must be separately optimized, thus limiting their use. Here we develop efficient and versatile systems, based on in vitro transcribed single-guide-RNAs (sgRNAs) and fluorescently-tagged recombinant, catalytically-inactivated Cas9 (dCas9) proteins. Controlled cell delivery of pre-assembled dCas9-sgRNA ribonucleoprotein (RNP) complexes enables robust genomic imaging in live cells and in early mouse embryos. We further demonstrate multiplex tagging of up to 3 genes, tracking detailed movements of chromatin segments and imaging spatial relationships between a distal enhancer and a target gene, with nanometer resolution in live cells. This simple and effective approach should facilitate visualizing chromatin dynamics and nuclear architecture in various living systems.

scholarly journals Simple and versatile imaging of genomic loci in live mammalian cells and early pre-implantation embryos using CAS-LiveFISH

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yongtao Geng ◽  
Alexandros Pertsinidis
Keyword(s):  
Mammalian Cells ◽  
Target Gene ◽  
Nuclear Architecture ◽  
Live Cells ◽  
Transcription Activator ◽  
Distal Enhancer ◽  
Chromatin Dynamics ◽  
Guide Rnas ◽  
Early Mouse

AbstractVisualizing the 4D genome in live cells is essential for understanding its regulation. Programmable DNA-binding probes, such as fluorescent clustered regularly interspaced short palindromic repeats (CRISPR) and transcription activator-like effector (TALE) proteins have recently emerged as powerful tools for imaging specific genomic loci in live cells. However, many such systems rely on genetically-encoded components, often requiring multiple constructs that each must be separately optimized, thus limiting their use. Here we develop efficient and versatile systems, based on in vitro transcribed single-guide-RNAs (sgRNAs) and fluorescently-tagged recombinant, catalytically-inactivated Cas9 (dCas9) proteins. Controlled cell delivery of pre-assembled dCas9-sgRNA ribonucleoprotein (RNP) complexes enables robust genomic imaging in live cells and in early mouse embryos. We further demonstrate multiplex tagging of up to 3 genes, tracking detailed movements of chromatin segments and imaging spatial relationships between a distal enhancer and a target gene, with nanometer resolution in live cells. This simple and effective approach should facilitate visualizing chromatin dynamics and nuclear architecture in various living systems.

scholarly journals Construction of adenovirus vectors simultaneously expressing four multiplex, double-nicking guide RNAs of CRISPR/Cas9 and in vivo genome editing

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tomoko Nakanishi ◽  
Aya Maekawa ◽  
Mariko Suzuki ◽  
Hirotaka Tabata ◽  
Kumiko Sato ◽  
...  
Keyword(s):  
Target Gene ◽  
Animal Experiments ◽  
Adenovirus Vector ◽  
Mouse Embryonic Fibroblast ◽  
Newborn Mouse ◽  
Guide Rnas ◽  
One Step ◽  
Target Effect

AbstractSimultaneous expression of multiplex guide RNAs (gRNAs) is valuable for knockout of multiple genes and also for effective disruption of a gene by introducing multiple deletions. We developed a method of Tetraplex-guide Tandem for construction of cosmids containing four and eight multiplex gRNA-expressing units in one step utilizing lambda in vitro packaging. Using this method, we produced an adenovirus vector (AdV) containing four multiplex-gRNA units for two double-nicking sets. Unexpectedly, the AdV could stably be amplified to the scale sufficient for animal experiments with no detectable lack of the multiplex units. When the AdV containing gRNAs targeting the H2-Aa gene and an AdV expressing Cas9 nickase were mixed and doubly infected to mouse embryonic fibroblast cells, deletions were observed in more than 80% of the target gene even using double-nicking strategy. Indels were also detected in about 20% of the target gene at two sites in newborn mouse liver cells by intravenous injection. Interestingly, when one double-nicking site was disrupted, the other was simultaneously disrupted, implying that two genes in the same cell may simultaneously be disrupted in the AdV system. The AdVs expressing four multiplex gRNAs could offer simultaneous knockout of four genes or two genes by double-nicking cleavages with low off-target effect.

scholarly journals A Proteomic Analysis of the Effects of Yessotoxin and Di-Desulfoyessotoxin on Yeast and Mammalian Cells

2021 ◽  
Author(s):  
◽  
Sarah Cordiner
Keyword(s):  
Cell Morphology ◽  
Hepg2 Cells ◽  
Mammalian Cells ◽  
Live Cells ◽  
Shellfish Poisoning ◽  
Hl60 Cells ◽  
Apoptosis Inducer ◽  
Blood Leukocyte ◽  
Shock Proteins

<p>Yessotoxin (YTX) is a disulfated polycyclic polyether, produced by dinoflagellate algae. It is known to accumulate in edible shellfish, raising concerns about its potential risk to human health. YTX was initially classified as a diarrhetic shellfish poisoning toxin, due to commonly being extracted alongside toxins of this variety. However, YTX does not induce any of the effects characteristic of this group. A separate category for YTXs was established by the European Commission in 2002 and a limit of 1 mg/kg of shellfish meat was established. YTX has been shown to be an apoptosis inducer in a variety of cell lines in vitro. It has also been shown to be lethal to mice when administered by intra-peritoneal injection. However, when administered orally only limited toxicity is observed. The di-desulfated derivative (dsYTX) has also been shown to be lethal to mice following intra-peritoneal injection. However it causes damage mainly to the liver, whereas YTX appears to target the heart. The mechanism of action of YTX is still unknown. The goals of this project were to use proteomic techniques, to examine the effects of YTX and dsYTX on Saccharomyces cerevisiae and human promyelocytic leukemic blood leukocyte (HL60) cells. Young et al. (2009) showed that the major proteins affected by YTX in HepG2 cells were heterogeneous ribonucleoproteins (hnRNPs), lamins, cathepsins and heat shock proteins. HnRNPs had not previously been identified as possible targets of YTX. Alterations of hnRNP levels were also seen in HL60 cells treated with microtubule stabilising agents, peloruside A or paclitaxel (Wilmes et al., 2011, 2012). No differences in cell morphology or significant changes in protein abundance were observed when S. cerevisiae cells were exposed to YTX. A small number of significant changes in abundance were detected when these cells were exposed to dsYTX. The small number of protein changes seen is possibly due to poor toxin entrance into the cell through the yeast cell wall, lack of protein targets structurally homologous to those found in mammalian cells, or fast removal of the toxin through export pumps. Twenty-four hour incubation of HL60 cells with YTX resulted in increased cell death but no change in cell morphology. Treatment with dsYTX caused cells to aggregate into clusters and a 24% decrease in the number of live cells. Increases were found in the abundance of β-actin, hnRNP A and BiP proteins in response to dsYTX treatment. Decreases in these proteins were seen in HepG2 cells treated with YTX for 24 hours. As seen in S. cerevisiae cells, dsYTX had a greater effect in HL60 cells compared with YTX. Overall, the results provide some support for the previously identified effect on hnRNPs in mammalian cells exposed to YTX.</p>

scholarly journals A Proteomic Analysis of the Effects of Yessotoxin and Di-Desulfoyessotoxin on Yeast and Mammalian Cells

2021 ◽  
Author(s):  
◽  
Sarah Cordiner
Keyword(s):  
Cell Morphology ◽  
Hepg2 Cells ◽  
Mammalian Cells ◽  
Live Cells ◽  
Shellfish Poisoning ◽  
Hl60 Cells ◽  
Apoptosis Inducer ◽  
Blood Leukocyte ◽  
Shock Proteins

<p>Yessotoxin (YTX) is a disulfated polycyclic polyether, produced by dinoflagellate algae. It is known to accumulate in edible shellfish, raising concerns about its potential risk to human health. YTX was initially classified as a diarrhetic shellfish poisoning toxin, due to commonly being extracted alongside toxins of this variety. However, YTX does not induce any of the effects characteristic of this group. A separate category for YTXs was established by the European Commission in 2002 and a limit of 1 mg/kg of shellfish meat was established. YTX has been shown to be an apoptosis inducer in a variety of cell lines in vitro. It has also been shown to be lethal to mice when administered by intra-peritoneal injection. However, when administered orally only limited toxicity is observed. The di-desulfated derivative (dsYTX) has also been shown to be lethal to mice following intra-peritoneal injection. However it causes damage mainly to the liver, whereas YTX appears to target the heart. The mechanism of action of YTX is still unknown. The goals of this project were to use proteomic techniques, to examine the effects of YTX and dsYTX on Saccharomyces cerevisiae and human promyelocytic leukemic blood leukocyte (HL60) cells. Young et al. (2009) showed that the major proteins affected by YTX in HepG2 cells were heterogeneous ribonucleoproteins (hnRNPs), lamins, cathepsins and heat shock proteins. HnRNPs had not previously been identified as possible targets of YTX. Alterations of hnRNP levels were also seen in HL60 cells treated with microtubule stabilising agents, peloruside A or paclitaxel (Wilmes et al., 2011, 2012). No differences in cell morphology or significant changes in protein abundance were observed when S. cerevisiae cells were exposed to YTX. A small number of significant changes in abundance were detected when these cells were exposed to dsYTX. The small number of protein changes seen is possibly due to poor toxin entrance into the cell through the yeast cell wall, lack of protein targets structurally homologous to those found in mammalian cells, or fast removal of the toxin through export pumps. Twenty-four hour incubation of HL60 cells with YTX resulted in increased cell death but no change in cell morphology. Treatment with dsYTX caused cells to aggregate into clusters and a 24% decrease in the number of live cells. Increases were found in the abundance of β-actin, hnRNP A and BiP proteins in response to dsYTX treatment. Decreases in these proteins were seen in HepG2 cells treated with YTX for 24 hours. As seen in S. cerevisiae cells, dsYTX had a greater effect in HL60 cells compared with YTX. Overall, the results provide some support for the previously identified effect on hnRNPs in mammalian cells exposed to YTX.</p>

scholarly journals High Mobility Group 1 Protein Is Not Stably Associated with the Chromosomes of Somatic Cells

1997 ◽  
Vol 137 (1) ◽  
pp. 19-26 ◽  
Author(s):  
Luca Falciola ◽  
Fabio Spada ◽  
Sabina Calogero ◽  
Gernot Längst ◽  
Renate Voit ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cultured Cells ◽  
High Mobility ◽  
Histone H1 ◽  
Biochemical Properties ◽  
High Mobility Group ◽  
Live Cells ◽  
Differentiated Cells ◽  
Group 1

High mobility group 1 (HMG1) protein is an abundant and conserved component of vertebrate nuclei and has been proposed to play a structural role in chromatin organization, possibly similar to that of histone H1. However, a high abundance of HMG1 had also been reported in the cytoplasm and on the surface of mammalian cells. We conclusively show that HMG1 is a nuclear protein, since several different anti-HMG1 antibodies stain the nucleoplasm of cultured cells, and epitope-tagged HMG1 is localized in the nucleus only. The protein is excluded from nucleoli and is not associated to specific nuclear structures but rather appears to be uniformly distributed. HMG1 can bind in vitro to reconstituted core nucleosomes but is not stably associated to chromatin in live cells. At metaphase, HMG1 is detached from condensed chromosomes, contrary to histone H1. During interphase, HMG1 readily diffuses out of nuclei after permeabilization of the nuclear membranes with detergents, whereas histone H1 remains associated to chromatin. These properties exclude a shared function for HMG1 and H1 in differentiated cells, in spite of their similar biochemical properties. HMG1 may be stably associated only to a very minor population of nucleosomes or may interact transiently with nucleosomes during dynamic processes of chromatin remodeling.

Cardiac transgenesis with the tetracycline transactivator changes myocardial function and gene expression

2005 ◽  
Vol 22 (1) ◽  
pp. 118-126 ◽  
Author(s):  
Diana T. McCloskey ◽  
Lynne Turnbull ◽  
Philip M. Swigart ◽  
Alexander C. Zambon ◽  
Sally Turcato ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Target Gene ◽  
Control Group ◽  
Transcription Activator ◽  
Tet System ◽  
Double Transgenic Mice ◽  
Regulated Gene Expression

The cardiac-specific tetracycline-regulated gene expression system (tet-system) is a powerful tool using double-transgenic mice. The cardiac α-myosin heavy chain promoter (αMHC) drives lifetime expression of a tetracycline-inhibited transcription activator (tTA). Crossing αMHC-tTA mice with mice containing a tTA-responsive promoter linked to a target gene yields double-transgenic mice having tetracycline-repressed expression of the target gene in the heart. Using the tet-system, some studies use nontransgenic mice for the control group, whereas others use single-transgenic αMHC-tTA mice. However, previous studies found that high-level expression of a modified activator protein caused cardiomyopathy. Therefore, we tested whether cardiac expression of tTA was associated with altered function of αMHC-tTA mice compared with wild-type (WT) littermates. We monitored in vivo and in vitro function and gene expression profiles for myocardium from WT and αMHC-tTA mice. Compared with WT littermates, αMHC-tTA mice had a greater heart-to-body weight ratio (≈10%), ventricular dilation, and decreased ejection fraction, suggesting mild cardiomyopathy. In vitro, submaximal contractions were greater compared with WT and were associated with greater myofilament Ca2+ sensitivity. Gene expression profiling revealed that the expression of 153 genes was significantly changed by >20% when comparing αMHC-tTA with WT myocardium. These findings demonstrate that introduction of the αMHC-tTA construct causes significant effects on myocardial gene expression and major functional abnormalities in vivo and in vitro. For studies using the tet-system, these results suggest caution in the use of controls, since αMHC-tTA myocardium differs appreciably from WT. Furthermore, the results raise the possibility that the phenotype conferred by a target gene may be influenced by the modified genetic background of αMHC-tTA myocardium.

scholarly journals Direct Observation of Sophorolipid Micelle Docking in Model Membranes and Cells by Single Particle Studies Reveals Optimal Fusion Conditions

Biomolecules ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1291 ◽  
Author(s):  
Pradeep Kumar Singh ◽  
Søren S.-R. Bohr ◽  
Nikos S. Hatzakis
Keyword(s):  
Cancer Cells ◽  
Hela Cells ◽  
Single Particle ◽  
Mammalian Cells ◽  
Model Membranes ◽  
Live Cells ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Sophorolipids (SLs) are naturally produced glycolipids that acts as drug delivery for a spectrum of biomedical applications, including as an antibacterial antifungal and anticancer agent, where they induce apoptosis selectively in cancerous cells. Despite their utility, the mechanisms underlying their membrane interactions, and consequently cell entry, remains unknown. Here, we combined a single liposome assay to observe directly and quantify the kinetics of interaction of SL micelles with model membrane systems, and single particle studies on live cells to record their interaction with cell membranes and their cytotoxicity. Our single particle readouts revealed several repetitive docking events on individual liposomes and quantified how pH and membrane charges, which are known to vary in cancer cells, affect the docking of SL micelles on model membranes. Docking of sophorolipids micelles was found to be optimal at pH 6.5 and for membranes with −5% negatively charge lipids. Single particle studies on mammalian cells reveled a two-fold increased interaction on Hela cells as compared to HEK-293 cells. This is in line with our cell viability readouts recording an approximate two-fold increased cytotoxicity by SLs interactions for Hela cells as compared to HEK-293 cells. The combined in vitro and cell assays thus support the increased cytotoxicity of SLs on cancer cells to originate from optimal charge and pH interactions between membranes and SL assemblies. We anticipate studies combining quantitative single particle studies on model membranes and live cell may reveal hitherto unknown molecular insights on the interactions of sophorolipid and additional nanocarriers mechanism.

scholarly journals Protein Interactions of the MLL PHD Fingers Modulate MLL Target Gene Regulation in Human Cells

2001 ◽  
Vol 21 (10) ◽  
pp. 3589-3597 ◽  
Author(s):  
Keri Fair ◽  
Melanie Anderson ◽  
Elena Bulanova ◽  
Huaifeng Mi ◽  
Maximilian Tropschug ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Protein Interactions ◽  
Mammalian Cells ◽  
Target Gene ◽  
Target Genes ◽  
Leukemia Cells ◽  
Altered Expression ◽  
Amino Acid Sequence Conservation

ABSTRACT The PHD fingers of the human MLL and Drosophila trx proteins have strong amino acid sequence conservation but their function is unknown. We have determined that these fingers mediate homodimerization and binding of MLL to Cyp33, a nuclear cyclophilin. These two proteins interact in vitro and in vivo in mammalian cells and colocalize at specific nuclear subdomains. Overexpression of the Cyp33 protein in leukemia cells results in altered expression ofHOX genes that are targets for regulation by MLL. These alterations are suppressed by cyclosporine and are not observed in cell lines that express a mutant MLL protein without PHD fingers. These results suggest that binding of Cyp33 to MLL modulates its effects on the expression of target genes.

scholarly journals Rapid nucleus-scale reorganization of chromatin in neurons enables transcriptional adaptation for memory consolidation

2020 ◽  
Author(s):  
Manuel Peter ◽  
Dominik F. Aschauer ◽  
Renata Vaz Pandolfo ◽  
Anne Sinning ◽  
Florian Grössl ◽  
...  
Keyword(s):  
Expression Patterns ◽  
Nuclear Architecture ◽  
Fast Time ◽  
Active Chromatin ◽  
Chromatin Dynamics ◽  
Transgenic Mouse Line ◽  
Transcriptional Adaptation ◽  
Chromatin Reorganization

AbstractThe interphase nucleus is functionally organized in active and repressed territories defining the transcriptional status of the cell. However, it remains poorly understood how the nuclear architecture of neurons adapts in response to behaviorally relevant stimuli that trigger fast alterations in gene expression patterns. Imaging of fluorescently tagged nucleosomes revealed that pharmacological manipulation of neuronal activity in vitro and auditory cued fear conditioning in vivo induce nucleus-scale restructuring of chromatin within minutes. Furthermore, the acquisition of auditory fear memory is impaired after infusion of a drug into auditory cortex which blocks chromatin reorganization in vitro. We propose that active chromatin movements at the nucleus scale act together with local gene-specific modifications to enable transcriptional adaptations at fast time scales. Introducing a transgenic mouse line for photolabeling of histones, we extend the realm of systems available for imaging of chromatin dynamics to living animals.

scholarly journals CAP1 binds and activates adenylyl cyclase in mammalian cells

2021 ◽  
Vol 118 (24) ◽  
pp. e2024576118
Author(s):  
Xuefeng Zhang ◽  
Alejandro Pizzoni ◽  
Kyoungja Hong ◽  
Nyla Naim ◽  
Chao Qi ◽  
...  
Keyword(s):  
Adenylyl Cyclase ◽  
Mammalian Cells ◽  
Feedback Loop ◽  
Critical Role ◽  
Small Gtpase ◽  
Actin Dynamics ◽  
Live Cells ◽  
Dependent Manner ◽  
Downstream Effector

CAP1 (Cyclase-Associated Protein 1) is highly conserved in evolution. Originally identified in yeast as a bifunctional protein involved in Ras-adenylyl cyclase and F-actin dynamics regulation, the adenylyl cyclase component seems to be lost in mammalian cells. Prompted by our recent identification of the Ras-like small GTPase Rap1 as a GTP-independent but geranylgeranyl-specific partner for CAP1, we hypothesized that CAP1-Rap1, similar to CAP-Ras-cyclase in yeast, might play a critical role in cAMP dynamics in mammalian cells. In this study, we report that CAP1 binds and activates mammalian adenylyl cyclase in vitro, modulates cAMP in live cells in a Rap1-dependent manner, and affects cAMP-dependent proliferation. Utilizing deletion and mutagenesis approaches, we mapped the interaction of CAP1-cyclase with CAP’s N-terminal domain involving critical leucine residues in the conserved RLE motifs and adenylyl cyclase’s conserved catalytic loops (e.g., C1a and/or C2a). When combined with a FRET-based cAMP sensor, CAP1 overexpression–knockdown strategies, and the use of constitutively active and negative regulators of Rap1, our studies highlight a critical role for CAP1-Rap1 in adenylyl cyclase regulation in live cells. Similarly, we show that CAP1 modulation significantly affected cAMP-mediated proliferation in an RLE motif–dependent manner. The combined study indicates that CAP1-cyclase-Rap1 represents a regulatory unit in cAMP dynamics and biology. Since Rap1 is an established downstream effector of cAMP, we advance the hypothesis that CAP1-cyclase-Rap1 represents a positive feedback loop that might be involved in cAMP microdomain establishment and localized signaling.

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