Los autores agradecen al prof. Chiara Campanella y AMRA Centro de Competencia de la Universidad de Nápoles Federico II, Nápoles, Italia, para darles el uso de la Microdisector láser, y el Sr. Vincenzo Vicidomini por la generosa ayuda de la animación 3D.

Parte 1. Preparación de los discos imaginal del ala de Drosophila Sometido a RNAi específico y localizado. Como material biológico, que utiliza discos imaginal del ala de Drosophila obtenida a partir de larvas transgénicas sometidas a la silenciamiento de los genes localizados y específicos por medio del sistema de GAL4/UAS 1. Esta progenie larval orginates de un cruce genético asociado con las dos líneas parentales: uno que lleva el conductor GAL4, la segunda la respuesta UAS. Además de expresar GAL4 en el patrón de un temporal específica y / o espaciales, la línea conductor lleva un reportero UAS-GFP que permite visualizar el dominio de la expresión GAL4. La línea de UAS de respuesta expresa, bajo el control de la secuencia UAS, una horquilla de silenciamiento del ARN (hpRNA) capaces de dirigirse específicamente a los genes seleccionados por su interés (lo llaman gen X) 2. Una vez que se expresa en la célula, el X hpRNA se escinde para generar pequeños ARN de interferencia (siRNA) capaz de silenciar específicamente el gen seleccionado. En la descendencia de larvas, que lleva el conductor y los transgenes de respuesta, es sólo la construcción UAS-silenciar transcripción en las células que expresan la proteína GAL4, y por lo tanto es capaz de inducir un silenciamiento restricción espacial de los genes seleccionados X. Entre la variedad de posibles aplicaciones, se ha aplicado este método para silenciar un gen seleccionado de nuestro interés (gen X) bajo el control de la engrailed-GAL4 (es) del conductor, que puede provocar el silenciamiento específico en el compartimento posterior del disco de ala 3 , cuyo territorio se caracterizó por la expresión del transgén UAS-GFP. Los discos fueron silenciados imaginal de ala mano disecada, teniendo cuidado de eliminar la contaminación de los tejidos circundantes, especialmente en la tráquea adheridos. Cada disco ala aislada fue entonces rápidamente y se transfieren cuidadosamente a un tratamiento previo, el marco disección de RNasa libre de microdisección láser inmediata de las áreas seleccionadas. Parte 2. Microdisección con láser de las áreas seleccionadas de los silenciados (posterior) y sin silenciador (anterior) compartimentos del disco del ala. Una vez que el disco estaba en el ala de la membrana, microdisección láser de las áreas específicas de silencio (posterior; GFP-etiquetado) y sin silenciador (anterior, GFP-etiquetados) compartimentos se logró. Este dibujo fue realizado por un área que podría encajar fácilmente en tanto, los lados positivos y las buenas prácticas agrarias-GFP-negativos del disco. Bajo el Microdisector, lo primero que se centró el rayo láser en el tejido GFP-positivas, haciendo un corte a lo largo del perímetro seleccionado. El producto Microdisector con corte a la velocidad seleccionada y el poder, pero es posible perfeccionar el corte de forma manual, hasta que el área seleccionada del tejido cae en el tubo por debajo. Este tubo contiene una pequeña cantidad del reactivo TRI, por lo que el tejido GFP-positivo está listo para la posterior extracción de RNA. Estamos posteriormente se trasladó al área de corte de lo contrario, GFP-negativos lado del disco de ala, con el fin de diseccionar un territorio equivalente a la sin silenciador, GFP-negativos tejidos. Una vez más, después de que el corte automático, se procedió a refinar el corte manual. Una vez que el corte se llevó a cabo, también el tejido GFP-negativos se recuperó en el reactivo TRI, listo para la extracción de RNA. Parte 3. La extracción de RNA y perfiles de transcripción de las áreas de tejido microdissected. Giramos los tubos para separar el líquido de la tapa y añade reactivo TRI hasta 1 ml, y luego realizaron la extracción de ARN, siguiendo el protocolo reactivo TRI estándar. Para recoger el material suficiente, se repitió el procedimiento de corte de 100 discos imaginal del ala. A partir de 100 zonas de alrededor de 5.000 m 2 cada uno, nuestro rendimiento fue de 1,5 g de ARN. Exactitud de la disección manual fue controlada entonces por comprobar la expresión de GFP en las muestras de silencio y sin silenciador por RT-PCR, usando Super Guión III (Invitrogen) para sintetizar el cDNA con hexámeros aleatorios. Posterior análisis cuantitativo se centró en determinar los niveles de expresión de los genes silenciados X, junto con los de supuestos objetivos de su vía de reglamentación, se llevaron a cabo por Real Time RT-PCR utilizando Master Mix (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>

Con respecto a sus niveles de expresión basal logrado en los compartimentos anterior / posterior de los discos del ala sin silenciador, la actividad del gen X seleccionados se encuentran reducidos al 40% en silencio. Por el contrario, uno de sus supuestos objetivos (genes Y) se encontró significativamente hasta reguladas (7 veces de aumento), que nos lleva a validar la hipótesis de que fue regulada negativamente por el gen X. Llegamos a la conclusión de que el enfoque descrito anteriorme...

Volar cepas Drosophila UAS-silenciar líneas se pueden obtener de VDRC RNAi colección 2. Una colección de líneas GAL4-driver está disponible en el Bloomington Drosophila Stock Center (Indiana, EE.UU.). Equipo El equipo requerido incluye láser Microdisector (Leica LMD6000), Coulter Microfuge 22R (Beckman), PCR (BIO-RAD Mi termociclador), PCR en Tiempo Real (BIO-RAD iQ5). Instrumentos Herramientas necesarias incluyen: pozos de vidrio, cepillo, pinzas de microdisección, colgando diapositivas caída, alfileres de insectos montados sobre agujas de jeringas, los marcos de metal con una membrana de PET, tubos de plástico (50 ml, 05 ml, 0,25 ml).

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

Naturaleza heterogénea de los tejidos ha demostrado ser un factor limitante en la cantidad de información que puede ser generada a partir de muestras biológicas, lo que compromete los análisis. Teniendo en cuenta las asociaciones celulares complejos y dinámicos que existen en muchos tejidos, con el fin de recapitular las interacciones in vivo el análisis molecular a fondo uno debe ser capaz de analizar poblaciones específicas de células dentro de su contexto de origen. Laser mediada por microdisección puede lograr este objetivo, lo que permite identificar de forma inequívoca y exitosa cosecha de las células de interés bajo visualización microscópica directa, manteniendo la integridad molecular. Hemos aplicado esta tecnología para analizar la expresión de genes dentro de áreas definidas del desarrollo del disco ala de Drosophila, lo que representa un modelo de sistema ventajoso para estudiar el control del crecimiento, la diferenciación celular y la organogénesis. Larval discos imaginales se subdivide en compartimentos precozmente anterior y posterior, dorsal y ventral de los límites de restricción de linaje. Haciendo uso de la inducible GAL4-UAS sistema de expresión binaria, cada uno de estos compartimentos pueden ser etiquetados específicamente en las moscas transgénicas que expresan un transgén UAS-GFP bajo el control de la adecuada GAL4 conductor de construcción. En los discos de transgénicos, perfiles de expresión génica de los subconjuntos de células discretas precisamente puede determinarse después de láser mediada por microdisección, utilizando la señal de GFP fluorescentes para guía de corte por láser. Entre la variedad de aplicaciones posteriores, nos centramos en perfiles de transcripción del ARN después de localizada la interferencia de ARN (RNAi). Con el advenimiento de la tecnología del RNAi, las buenas prácticas agrarias de etiquetado puede ir acompañado de caída localizada de un gen determinado, lo que permite determinar la respuesta transcripcional de una población de células discretas para el silenciamiento de genes específicos. Para validar este enfoque, hemos diseccionado áreas equivalentes del disco desde la parte posterior (marcado por la expresión de GFP), y la anterior (sin etiqueta) en compartimiento de silenciar regional en el compartimiento de P de un gen de otro modo de expresión ubicua. Se extrajo el ARN de microdissected áreas silenciadas y sin silenciador y el perfil comparativo de la expresión de genes determinados por cuantitativa en tiempo real RT-PCR. Se demuestra que este método se puede aplicar con eficacia para la transcriptómica precisa de los subconjuntos de células dentro de los discos imaginales de Drosophila. En efecto, mientras que la preparación del disco masiva como fuente de ARN en general, supone homogeneidad de la célula, es bien sabido que la expresión de la transcripción puede variar mucho dentro de estas estructuras como consecuencia de la información de posición. Utilizando la señal fluorescente GFP localizada a guía de corte por láser, los análisis de la transcripción más precisa se puede realizar y rentable aplicado a diferentes aplicaciones, incluyendo perfiles de transcripción de los distintos linajes de células dentro de su contexto de origen.

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

Microdisección láser se aplicó para analizar perfiles de expresión génica en compartimentos específicos de Drosophila disco ala sometida a localizado RNAi<em&gt; In vivo</em&gt;. ARN extraído de las áreas equivalentes de los compartimentos de silencio y sin silenciador fue analizada por RT-PCR cuantitativa para determinar perfiles de expresión genética comparada en el contexto de microecología tejido nativo.

Microdisección láser aplicada a perfiles de expresión génica de subconjunto de las células de la Drosophila Ala Disco

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 vistas.  University of Naples. Microdisección láser se aplicó para analizar perfiles de expresión génica en compartimentos específicos de Drosophila disco ala sometida a localizado RNAi In vivo. ARN extraído de las áreas equivalentes de los compartimentos de silencio y sin silenciador fue analizada por RT-PCR cuantitativa para determinar perfiles de expresión genética comparada en el contexto de microecología tejido nativo.

Video: 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.

Láser microdisección de captura de tejidos de mamíferos

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

Investigación

Biología

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 preparación de cultivos primarios de células hematopoyéticas de la médula ósea murina por electroporación

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.

Utilizando el sistema de electroporación Gene Emisores MXcell para transfectar células primarias con alta eficiencia

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.

El uso de un contador de células automatizado para simplificar estudios de expresión génica: siRNA Derribo de IL-4 la expresión génica en células dependientes 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.

Las secciones incluidas en parafina y congeladas de Drosophila Los músculos de Adultos

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.

En la reconstitución in vitro de los activos T. castaneum telomerasa

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.

Aislamiento a base de afinidad de Tagged Núcleos de<em&gt; Drosophila</em&gt; Los tejidos para análisis de expresión génica

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.

Análisis de RNA Global Síntesis en el nivel de células individuales siguiente Hipoxia

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.

Screening siRNA para identificar ubiquitina y reguladores del sistema ubiquitina-como de las vías biológicas en células de mamífero en cultivo

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.