Ce travail a été financé en partie par la National Science Foundation (IOS-0920417) (ACL).

Aucun conflit d&#39;intérêt déclaré.

1. Modifications au Protocole actuel 1,1 appareils d&#39;enregistrement et de considérations débit En raison de sa disponibilité commerciale, la LumiCycle (Actimetrics) est devenu l&#39;appareil le plus couramment utilisé luminomètre automatisé pour enregistrement en temps réel 4,5,9,19,29-31. Le LumiCycle emploie des tubes photomultiplicateurs (PMT) en tant que détecteurs de lumière, qui offrent une extrême sensibilité et faible bruit 14,</...

<table border="1"><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> DMEM </td><td> HyClone </td><td> SH30243FS </td><td> Pour la croissance cellulaire régulier </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 12100-046 </td><td> Pour luminométrie </td></tr><tr><td> FBS </td><td> HyClone </td><td> SH3091003 </td><td></td></tr><tr><td> Pen / Strep / Gln (100x) </td><td> HyClone </td><td> SV3008201 </td><td></td></tr><tr><td> B-27 </td><td> Invitrogen </td><td> 17504-044 </td><td></td></tr><tr><td> D-luciférine </td><td> Biosynth </td><td> L-8220 </td><td></td></tr><tr><td> La poly-L-lysine </td><td> Sigma </td><td> P4707 </td><td></td></tr><tr><td> Polybrène </td><td> Millipore </td><td&gt; TR-1003-G </td><td></td></tr><tr><td> La forskoline </td><td> Sigma </td><td> F6886 </td><td></td></tr><tr><td> Tous les autres produits chimiques </td><td> Sigma </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> Équipement </td></tr><tr><td> Incubateur de culture tissulaire </td><td></td><td></td><td> 5% de CO2 à 37 ° C </td></tr><tr><td> Hotte de culture de tissus </td><td></td><td></td><td> BSL-2 certifié </td></tr><tr><td> Lumière fluorescente et microscope </td><td></td><td></td><td> Contraste de phase optionnelle </td></tr><tr><td> LumiCycle </td><td> Actimetrics </td><td></td><td></td></tr></table>

monitoring cell-autonomous circadian clock rhythms of gene expression using luciferase bioluminescence reporters

Chez les mammifères, de nombreux aspects du comportement et de la physiologie comme cycles veille-sommeil et le métabolisme du foie sont régis par horloges circadiennes endogènes (revu 1,2). Le rythme circadien garde-temps est un système hiérarchique multi-oscillateur réseau, avec l&#39;horloge centrale située dans le noyau suprachiasmatique (NSC) la synchronisation et la coordination des horloges extra-SCN et périphérique ailleurs 1,2. Les cellules individuelles sont des unités fonctionnelles pour la production et l&#39;entretien des rythmes circadiens 3,4, et ces oscillateurs de différents types de tissus de la part d&#39;un organisme remarquablement semblable mécanisme biochimique rétroaction négative. Toutefois, en raison des interactions au niveau du réseau neuronal dans le SCN et à travers rythmiques, les indices systémiques au niveau organismique, les rythmes circadiens au niveau organismique ne sont pas nécessairement cell-autonomous 5-7. Par rapport aux études traditionnelles de l&#39;activité locomotrice in vivo et ex vivo d&#39;explants SCN, cell-fondé des tests in vitro permettent la découverte de cellules autonomes défauts circadiens 5,8. Stratégiquement, à base de cellules modèles sont plus dociles expérimentalement pour la caractérisation phénotypique et la découverte rapide des mécanismes d&#39;horloge de base 5,8-13. Parce que les rythmes circadiens sont dynamiques, les mesures longitudinales avec une haute résolution temporelle sont nécessaires pour évaluer la fonction horloge. Ces dernières années, l&#39;enregistrement bioluminescence en temps réel en utilisant la luciférase de luciole en tant que journaliste est devenu une technique courante pour étudier les rythmes circadiens chez les mammifères 14,15, car elle permet l&#39;examen de la persistance et de la dynamique moléculaire des rythmes. Pour surveiller des cellules autonomes rythmes circadiens de l&#39;expression des gènes, les journalistes luciférase peut être introduit dans les cellules par transfection transitoire 13,16,17 ou transduction stable 5,10,18,19. Nous décrivons ici un protocole de transduction stable à l&#39;aide de gènes médié par lentivirus. Te système de vecteur lentiviral est supérieur aux méthodes traditionnelles telles que la transfection transitoire et la transmission germinale en raison de son efficacité et la polyvalence: il permet l&#39;exécution efficace et une intégration stable dans le génome de l&#39;hôte à la fois la division et non les cellules en division 20. Une fois une lignée cellulaire journaliste est établie, la dynamique de la fonction d&#39;horloge peut être examiné à travers l&#39;enregistrement de bioluminescence. Nous décrivons d&#39;abord la génération de P (Per2)-d lignes journaliste Luc, puis présentent les données de cette étude et d&#39;autres journalistes circadiens. Dans ces essais, les fibroblastes de souris 3T3 et U2OS cellules d&#39;ostéosarcome humains sont utilisés comme modèles cellulaires. Nous discutons également de multiples façons d&#39;utiliser ces modèles dans les études d&#39;horloge circadienne. Les méthodes décrites ici peuvent être appliqués à une grande variété de types de cellules pour étudier les bases cellulaires et moléculaires des horloges circadiennes, et peut s&#39;avérer utile dans la résolution des problèmes dans d&#39;autres systèmes biologiques.

Watch this Scientific Journal Video about Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters at JoVE.com

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

Horloges circadiennes fonctionner dans des cellules individuelles, c&#39;est à dire qu&#39;ils sont des cellules autonomes. Ici, nous décrivons les méthodes pour générer des modèles d&#39;horloge cellules autonomes utilisant des méthodes non-invasives, la luciférase technologie basée sur la bioluminescence en temps réel. Cellules Reporter fournir traitables, des systèmes de modèles fonctionnels pour l&#39;étude de la biologie circadienne.

Surveillance de cellules autonomes Rythmes horloge circadienne de l&#39;expression génique par Reporters Bioluminescence luciférase

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

monitoring-cell-autonomous-circadian-clock-rhythms-gene-expression

Research

JoVE Journal

Biology

22.1K vues.  The University of Memphis. Horloges circadiennes fonctionner dans des cellules individuelles, c'est à dire qu'ils sont des cellules autonomes. Ici, nous décrivons les méthodes pour générer des modèles d'horloge cellules autonomes utilisant des méthodes non-invasives, la luciférase technologie basée sur la bioluminescence en temps réel. Cellules Reporter fournir traitables, des systèmes de modèles fonctionnels pour l'étude de la biologie circadienne.

Vidéo: Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

Bacterial Gene Expression Analysis Using Microarrays

Analyse bactérienne expression génique par puces à ADN

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 ...

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 ...

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 ...

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.

Mesure des taux métabolique dans<em&gt; Drosophila</em&gt; En utilisant Respirométrie

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for in vivo studies. Our lab has demonstrated that a modification to the carboxy-terminus of Gaussia luciferase confers secretion of the reporter in response to ER calcium depletion. The methods for using a luciferase based, secreted ER calcium monitoring protein (SERCaMP) for in vitro and in vivo applications are described herein. This video highlights hepatic injections, pharmacological manipulation of GLuc-SERCaMP, blood collection and processing, and assay parameters for longitudinal monitoring of ER calcium.

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

Suivi réticulum endoplasmique homéostasie calcique l&#39;aide d&#39;un<em&gt; Gaussia</em&gt; Luciférase SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

Détection de intracellulaire Gene Expression dans des cellules vivantes de murin, humain et porcin Origine utilisant des nanoparticules marquées par fluorescence

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable biotic and abiotic stress. Conversely, disruption of circadian timekeeping severely compromises plant health and reduces agricultural crop yields. It is therefore imperative that we understand the intricate regulation of circadian rhythms in plants, including the factors that affect motion of the transcriptional clockwork itself.
Testing circadian defects in the model plant Arabidopsis thaliana (Arabidopsis) traditionally involves crossing specific mutant lines to a line rhythmically expressing firefly luciferase from a circadian clock gene promoter. This approach is laborious, time-consuming, and could be fruitless if a mutant has no circadian phenotype. The methodology presented here allows a rapid initial assessment of circadian phenotypes. Protoplasts derived from mutant and wild-type Arabidopsis are isolated, transfected with a rhythmically expressed luminescent reporter, and imaged under constant light conditions for 5 days. Luminescent traces will directly reveal whether the free-running period of mutant plants is different from wild-type plants. The advantage of the method is that any Arabidopsis line can efficiently be screened, without the need for generating a stably transgenic luminescent clock marker line in that mutant background.

The circadian clock regulates about a third of the Arabidopsis transcriptome, but the percentage of genes that feed back into timekeeping remains unknown. Here we visualize a method to rapidly assess circadian phenotypes in any mutant line of Arabidopsis using luminescent imaging of a circadian reporter transiently expressed in protoplasts.

Analyse rapide des phénotypes circadiens dans Arabidopsis protoplastes transfectées avec un Luminescent Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable ...