Les auteurs remercient le Prof. Chiara Campanella et AMRA Centre de Compétence, Université de Naples Federico II de Naples, en Italie, pour leur fournir de l&#39;utilisation de la microdissecteur laser, et M. Vincenzo Vicidomini de l&#39;aide généreuse de l&#39;animation 3D.

Partie 1. Préparation de disques drosophile Wing Imaginal Soumis à l&#39;ARNi spécifiques et localisées. Comme matériel biologique, nous avons utilisé des disques imaginaux aile drosophile obtenue à partir de larves transgéniques soumises à l&#39;inactivation des gènes localisés et spécifiques par le biais du système GAL4/UAS 1. Cette descendance larvaire orginates d&#39;un croisement génétique impliquant deux lignées parentales: l&#39;un portant le pilote GAL4, le second le répondeur SAMU. En plus d&#39;exprimer GAL4 dans le modèle une temporels spécifiques et / ou spatiale, la ligne du pilote effectue un journaliste UAS-GFP qui permet de visualiser le domaine de GAL4 expression. La ligne de SAU répondeur exprime, sous le contrôle de la séquence UAS, une épingle RNA silencing (hpRNA) capable de cibler spécifiquement le gène d&#39;intérêt sélectionnés (appelons-le gène X) 2. Une fois exprimée dans la cellule, le X hpRNA est clivé pour générer de petits ARN interférents (siRNA) capable de taire les gènes spécifiquement sélectionnés. Dans la descendance des larves, qui transporte le conducteur et le répondeur transgènes, la construction SAMU-taire est seulement transcrit dans les cellules exprimant la protéine GAL4, et est donc capable d&#39;induire une silençage spatialement restreinte du gène sélectionné X. Parmi la variété des applications possibles, nous avons appliqué cette approche pour réduire au silence un gène choisi de notre intérêt (gène X) sous le contrôle de l&#39;engrailed-GAL4 (fr) conducteur, qui peut déclencher taire spécifique dans le compartiment postérieur du disque aile 3 , dont le territoire a été marquée par l&#39;expression du transgène UAS-GFP. Les disques ont été réduits au silence l&#39;aile imaginale main disséquée, en prenant soin d&#39;éliminer les tissus environnants contaminant, en particulier les trachées adhérents. Chaque disque aile isolée a ensuite été transféré rapidement et en douceur à un précédemment traités, cadre RNase dissection gratuit pour microdissection laser immédiate des zones sélectionnées. Partie 2. Microdissection laser des zones sélectionnées à partir du silence (postérieure) et unsilenced (antérieure) compartiments du disque aile. Une fois le disque d&#39;aile a été sur la membrane, microdissection laser des zones spécifiques du silence (postérieure; GFP-étiquetée) et unsilenced (antérieure; GFP-étiquetés) compartiments a été atteint. Ce dessin a été réalisé par une zone qui pourrait facilement s&#39;insérer dans les deux, les côtés positifs GFP et GFP-négatif du disque. Sous la microdissecteur, nous avons d&#39;abord concentré le faisceau laser sur les tissus GFP-positives, faisant une coupe le long du périmètre choisi. Le produit microdissecteur avec la coupe à la vitesse sélectionnée et le pouvoir, mais il est possible d&#39;affiner le découpage à la main, jusqu&#39;à ce que la zone sélectionnée de tissu tombe dans le tube en dessous. Ce tube contient une petite quantité de réactif TRI, de sorte que le tissu GFP-positives est prêt pour l&#39;ARN extraction ultérieure. Nous avons ensuite déplacé la zone de coupe à l&#39;opposé, la GFP-négatif côté du disque d&#39;aile, afin de disséquer un territoire équivalent de la unsilenced, GFP-négative des tissus. Une fois encore, après la coupe automatique, nous avons procédé à l&#39;affinage du coupé manuellement. Une fois que la coupe a été faite, aussi le tissu GFP-négative a été récupéré dans le réactif TRI, prêt pour l&#39;extraction de l&#39;ARN. Partie 3. Extraction d&#39;ARN et de profilage de transcription à partir des zones tissulaires microdisséquées. Nous filé les tubes de détacher le liquide de la PAC et a ajouté Tri Reagent jusqu&#39;à 1 ml, puis effectué l&#39;extraction d&#39;ARN suivant le protocole réactif TRI standard. Pour recueillir assez de matériel, nous avons répété la procédure de coupe sur les 100 disques imaginaux aile. A partir de 100 domaines d&#39;environ 5000 um 2 chacun, notre rendement a été de 1,5 ug d&#39;ARN. Précision de la dissection manuelle a ensuite été contrôlée en vérifiant expression de la GFP dans les échantillons réduits au silence et unsilenced par RT-PCR, en utilisant de Super Script III (Invitrogen) pour synthétiser l&#39;ADNc avec des hexamères aléatoires. Après analyses quantitatives consacrées à déterminer les niveaux d&#39;expression de la gène X silence, avec ceux de cibles putatives de sa voie réglementaire, ont été effectués par Real Time RT-PCR en utilisant Mix Master (Invitrogen).

<ol>
	<li>Elliott, D. A., Brand, A. H., Dahmann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+GAL4+System+A+Versatile+System+for+the+Expression+of+Gene.">The GAL4 System A Versatile System for the Expression of Gene.</a> , (2008).</li><li>Klein, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wing+disc+development+in+the+fly:+the+early+stages.+.">Wing disc development in the fly: the early stages. .</a> Current Opinion in Genetics &#38; Development. 11, 470-475 (2001).</li><li>Dietzl, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+transgenic+RNAi+library+for+conditional+gene+inactivation+in+Drosophila.">A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila.</a> Nature. 448, 151-U1-151-U1 (2007).</li><li>Martin, F. A., Morata, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartments+and+the+control+of+growth+in+the+Drosophila+wing+imaginal+disc.">Compartments and the control of growth in the Drosophila wing imaginal disc.</a> Development. 133, 4421-4426 (2006).</li><li>Tabata, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+morphogen+gradients.">Genetics of morphogen gradients.</a> Nature Reviews Genetics. 2, 620-630 (2001).</li><li>Moreno, E. &. a. m. p. ;. a. m. p., Basler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dMyc+transforms+cells+into+super-competitors.">dMyc transforms cells into super-competitors.</a> Cell. 117, 117-129 (2004).</li></ol>

En ce qui concerne leurs niveaux d&#39;expression basale réalisés dans les compartiments antérieur / postérieur de l&#39;aile unsilenced disques, l&#39;activité du gène X sélectionnés a été trouvée réduite à 40% à faire taire. En revanche, une de ses cibles putatives (gènes Y) a été trouvée significativement régulée à la hausse (7 fois-augmentation), nous conduit à valider l&#39;hypothèse qu&#39;il était régulé négativement par le gène X. Nous concluons que l&#39;...

Fly souches Drosophile SAMU-taire lignes peuvent être obtenus à partir de VDRC ARNi collecte 2. Une collection de GAL4-pilote de lignes sont disponibles au Centre de Bloomington drosophile Stock (Indiana, USA). Équipement L&#39;équipement nécessaire comprend laser microdissecteur (Leica LMD6000), Coulter Microfuge 22R (Beckman), PCR (BIO-RAD Mon Cycler), PCR en temps réel (BIO-RAD iQ5). Outils Les outils nécessaires sont: verre puits, brosse, pince microdissection, diapositives goutte suspendue, épingles insectes montés sur des aiguilles de seringues, des cadres métalliques à membrane en PET, tubes en plastique (50 ml, 05 ml, 0,25 ml).

laser microdissection applied to gene expression profiling of subset of cells from the drosophila wing disc

Nature hétérogène des tissus s&#39;est avérée être un facteur limitant dans la quantité d&#39;informations qui peuvent être générés à partir d&#39;échantillons biologiques, ce qui compromet les analyses en aval. Considérant les associations complexes et dynamiques cellulaires existant dans de nombreux tissus, afin de récapituler les interactions in vivo analyse approfondie moléculaires on doit être capable d&#39;analyser les populations cellulaires spécifiques au sein de leur contexte d&#39;origine. Laser à médiation microdissection pouvons atteindre cet objectif, permettant l&#39;identification non ambiguë et bonne récolte de cellules d&#39;intérêt sous visualisation microscopique direct tout en conservant l&#39;intégrité moléculaire. Nous avons appliqué cette technologie pour analyser l&#39;expression génique dans des zones définies du disque de développement chez la drosophile aile, ce qui représente un système modèle avantageux pour étudier le contrôle de croissance, la différenciation cellulaire et l&#39;organogenèse. Larves disques imaginaux sont précocement subdivisé en compartiments antérieur et postérieur, dorsale et ventrale par des frontières restriction lignée. Faisant usage de l&#39;inductible GAL4 UAS-système d&#39;expression binaire, chacun de ces compartiments peuvent être spécifiquement étiquetés mouches transgéniques exprimant un transgène UAS-GFP sous le contrôle du pilote approprié GAL4-construire. Dans les disques transgéniques, le profilage d&#39;expression génique de sous-ensembles distincts de cellules peut être précisément déterminé après microdissection laser médiation, en utilisant le signal GFP fluorescentes pour guider découpées au laser. Parmi la variété d&#39;applications en aval, nous nous sommes concentrés sur les profils de transcription ARN après localisée interférence ARN (ARNi). Avec l&#39;avènement de l&#39;ARNi de la technologie, la GFP étiquetage peut être couplé avec knockdown localisée d&#39;un gène donné, permettant pour déterminer la réponse transcriptionnelle d&#39;une population cellulaire discrète à l&#39;inactivation des gènes spécifiques. Afin de valider cette approche, nous disséqué zones équivalentes du disque de la partie postérieure (marqué par l&#39;expression de GFP), et la partie antérieure (sans étiquette) du compartiment à faire taire régionale dans le compartiment d&#39;un gène P contraire exprimée de manière ubiquitaire. L&#39;ARN a été extrait de microdissection des zones réduites au silence et unsilenced et comparative du profil d&#39;expression génique déterminé par quantitative en temps réel RT-PCR. Nous montrons que cette méthode peut être effectivement appliquée pour la transcriptomique précise des sous-ensembles de cellules au sein des disques imaginaux chez la drosophile. En effet, tandis que la préparation du disque massifs comme source d&#39;ARN suppose généralement l&#39;homogénéité des cellules, il est bien connu que l&#39;expression transcriptionnelle peut varier considérablement au sein de ces structures à la suite d&#39;informations de position. En utilisant le signal GFP fluorescentes localisées au guide de coupe au laser, plus précis analyses transcriptionnelles peuvent être réalisées et rentable appliqué à des applications disparates, y compris les profils de transcription des lignées cellulaires distinctes au sein de leur contexte d&#39;origine.

Watch this Scientific Journal Video about Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc at JoVE.com

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Microdissection laser a été appliquée pour analyser les profils d&#39;expression génique dans des compartiments spécifiques de la drosophile aile disque soumis à localisées ARNi<em&gt; In vivo</em&gt;. ARN extrait de zones équivalentes des compartiments silence et unsilenced a été analysée par RT-PCR quantitative pour déterminer comparative profil d&#39;expression génique dans le cadre de microécologie tissu natif.

Microdissection laser appliquée au profil d&#39;expression génique de la Sous-ensemble de cellules provenant de la Drosophile Wing disque

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, ...

laser-microdissection-applied-to-gene-expression-profiling-subset

Research

JoVE Journal

Biology

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

11.1K vues.  University of Naples. Microdissection laser a été appliquée pour analyser les profils d'expression génique dans des compartiments spécifiques de la drosophile aile disque soumis à localisées ARNi In vivo. ARN extrait de zones équivalentes des compartiments silence et unsilenced a été analysée par RT-PCR quantitative pour déterminer comparative profil d'expression génique dans le cadre de microécologie tissu natif.

Vidéo: Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser Capture Microdissection of Mammalian Tissue

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.

Microdissection laser des tissus de mammifères

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

Recherche

Biologie

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

La préparation des cultures primaires de cellules hématopoïétiques de la moelle osseuse murine pour l&#39;électroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Utilisation du système d&#39;électroporation Gene Pulser MXcell à transfecter des cellules primaires à haut rendement

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

Aide d&#39;un compteur de cellules automatisé pour simplifier les études d&#39;expression génique: Knockdown siRNA de l&#39;IL-4 dans l&#39;expression des gènes dépendant cellules Namalwa

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

Les articles inclus en paraffine et congelés de Drosophile</emMuscles adultes>

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

La reconstitution in vitro de l&#39;actif T. La télomérase castaneum

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene expression in these tissues. Thus, to analyze cell-specific gene expression profiles from Drosophila tissues, it may be necessary to isolate specific cell types with high purity and at sufficient yields for downstream applications such as transcriptional profiling and chromatin immunoprecipitation. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in these tissues, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). Here, an alternative approach to characterizing cell-specific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells, is described. Nuclei in the specific cell type of interest are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. The approach described in this protocol enables consistent isolation of nuclei from specific cell types in the Drosophila larval central nervous system at high purity and at sufficient levels for expression analysis, even when these cell types comprise less than 2% of the total cell population in the tissue. This approach can be used to isolate nuclei from a wide variety of Drosophila embryonic and larval cell types using specific Gal4 drivers, and may be useful for isolating nuclei from cell types that are not suitable for FACS or laser microdissection.

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila tissues often contain a heterogeneous mixture of cell types.&#160;To examine gene expression in specific cell types from a particular tissue, nuclei can be genetically tagged and subsequently isolated using an affinity-based approach. Isolated nuclei can be used for downstream applications such as gene expression analysis and chromatin immunoprecipitation.

Isolation basée sur l&#39;affinité de Tagged noyaux de<em&gt; Drosophila</em&gt; tissus pour l&#39;analyse de l&#39;expression génique

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Analyse de Global ARN de synthèse à la Cellule Plain suivante hypoxie

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

siRNA contrôle pour la ubiquitine et les régulateurs du système ubiquitine-comme des voies biologiques dans des cellules de mammifères en culture

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.