Os autores agradecem a prof. Chiara Campanella e AMRA Centro de Competência da Universidade de Nápoles Federico II, Nápoles, Itália, para proporcionar-lhes o uso do microdissector laser, e Vincenzo Vicidomini ajuda generosa na animação 3D.

Parte 1. Preparação de discos Drosophila Asa Imaginal Sujeitos a RNAi específica e localizada. Como material biológico, foram utilizados discos de Drosophila asa imaginal obtido a partir de larvas transgênicas submetidas ao silenciamento de genes localizados e específicos, por meio do sistema GAL4/UAS 1. Esta progênie larval orginates de um cruzamento genético envolvendo duas linhas parentais: uma carregando o driver GAL4, o segundo a responder UAS. Além de expressar GAL4 no padrão temporal específica e / ou espacial, a linha do driver carrega um repórter UAS-GFP, que permite visualizar a expressão de domínio GAL4. A linha de resposta UAS expressa, sob o controle da seqüência UAS, um hairpin silenciamento de RNA (hpRNA) capaz de visar especificamente o gene de interesse seleccionados (chamemos-lhe gene X) 2. Uma vez expresso na célula, o X hpRNA é clivada para gerar pequeno RNA de interferência (siRNA) capaz de silenciar o gene especificamente selecionados. Na progênie larval, que leva o condutor e responder transgenes, a construção UAS silenciar só é transcrito nas células que expressam a proteína GAL4 e, portanto, é capaz de induzir um silenciamento espacialmente restrita do gene selecionadas X. Entre a variedade de aplicações possíveis, aplicou-se esta abordagem para silenciar um gene selecionado de nosso interesse (gene X) sob o controle do engrailed-GAL4 (en) driver, que pode desencadear o silenciamento específico no compartimento posterior do disco ala de 3 , cujo território foi marcado pela expressão do transgene UAS-GFP. O silenciados discos asa imaginal mão-foram dissecados, tendo o cuidado de eliminar tecidos circundantes contaminação, especialmente a traquéia aderir. Cada disco ala isolada foi então rapidamente e delicadamente transferido para um, RNase previamente tratados quadro dissecção livre para microdissecção a laser imediata de áreas selecionadas. Parte 2. Microdissecção a laser de áreas selecionadas a partir do silêncio (posterior) e unsilenced (anterior) compartimentos do disco asa. Uma vez que o disco foi asa na membrana, microdissecção a laser de áreas específicas de silenciados (posterior; GFP-rotulados) e unsilenced (anterior; GFP-unlabelled) compartimentos foi alcançado. Este desenho foi realizado por uma área que poderia se encaixar facilmente em ambos, os lados positivos e GFP-GFP-negativos do disco. Sob a microdissector, primeiro foco do raio laser sobre o tecido GFP-positivo, fazendo um corte ao longo do perímetro selecionado. O produto microdissector com corte na velocidade selecionada e poder, mas é possível refinar o corte manualmente, até que a área selecionada de tecido cai no tubo embaixo. Este tubo contém um pequeno volume do Reagente TRI, de modo que o tecido GFP-positivo está pronto para a extração de RNA subseqüentes. Nós posteriormente mudou-se a área de corte para o oposto, GFP-negativos lado do disco asa, a fim de dissecar um território equivalente a partir da unsilenced, tecido GFP-negativos. Mais uma vez, após o corte automático, procedeu-se a refinar o corte manual. Uma vez que o corte foi feito, também o tecido GFP-negativos foi recuperada na Reagente TRI, pronto para a extração de RNA. Parte 3. A extração de RNA e perfil de transcrição de Áreas Tissue microdissecção. Nós girou os tubos para separar o líquido do cap e acrescentou Reagente TRI até 1 ml, então realizada extração de RNA, seguindo o protocolo padrão Reagente TRI. Para coletar material suficiente, nós repetimos o procedimento de corte em 100 discos de asa imaginal. A partir de 100 áreas de cerca de 5000 M 2 cada, nosso rendimento foi de 1,5 microgramas de RNA. Precisão da dissecção manual foi então controlado por verificação expressão GFP nas amostras silenciadas e unsilenced por RT-PCR, usando Super Script III (Invitrogen) para sintetizar cDNA com hexâmeros Random. Subseqüentes análises quantitativas focado para determinar os níveis de expressão do gene X silenciado, juntamente com os dos alvos putativos de sua via de regulamentação, foram realizadas por Real Time RT-PCR usando 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>

Em relação aos seus níveis de expressão basal alcançado nos compartimentos anterior / posterior da asa unsilenced discos, a atividade do gene X selecionada foi encontrado reduzidos para 40% sobre o silenciamento. Em contraste, um dos seus alvos putativos (genes Y) foi encontrada significativamente sobre-regulada (7 vezes maior), levando-nos para validar a hipótese de que ela foi regulada negativamente pelo gene X. Concluímos que a abordagem acima descrita experimental pode ser aplicad...

Fly cepas Drosophila UAS silenciando-linhas podem ser obtidas VDRC RNAi coleção de 2. Uma coleção de GAL4 driver-linhas está disponível no Drosophila Bloomington stock Center (Indiana, EUA). Equipamento Equipamentos necessários inclui Laser microdissector (Leica LMD6000), Coulter Microcentrifugue 22R (Beckman), PCR (BIO-RAD My Cycler), Real Time PCR (BIO-RAD iQ5). Ferramentas Ferramentas necessárias incluem: poços de vidro, escova, pinça de microdissecção, slides gotas de suspensão, alfinetes de insetos montados em agulhas de seringa, armações de metal com membrana 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

Heterogeneidade de tecidos tem provado ser um fator limitante na quantidade de informações que podem ser gerados a partir de amostras biológicas, comprometendo análises a jusante. Considerando-se as associações complexas e dinâmicas celulares existentes dentro muitos tecidos, a fim de recapitular a nas interações vivo análise minuciosa molecular é preciso ser capaz de analisar populações de células específicas dentro de seu contexto nativo. Laser mediada por microdissecção pode alcançar este objetivo, permitindo uma identificação inequívoca e colheita bem-sucedida de células de interesse sob visualização microscópica direta, mantendo a integridade molecular. Temos aplicado esta tecnologia para analisar a expressão de gene dentro das áreas definidas do desenvolvimento de Drosophila disco asa, o que representa um sistema modelo para estudar vantajoso controlar o crescimento, diferenciação celular e organogênese. Larval discos imaginais são precocemente subdividida em compartimentos anterior e posterior, dorsal e ventral por fronteiras restrição linhagem. Fazendo uso da induzível GAL4-UAS sistema de expressão de binário, cada um desses compartimentos podem ser rotulados especificamente em moscas transgênicos expressando um transgene UAS-GFP sob o controle do driver apropriado GAL4 construir. Nos discos transgênicos, perfil de expressão gênica de subconjuntos discretos de células pode ser determinada precisamente após laser-mediada microdissecção, usando o sinal fluorescente GFP para guia de corte a laser. Entre a variedade de aplicações a jusante, nos concentramos em RNA perfil transcrição localizada após interferência de RNA (RNAi). Com o advento da tecnologia de RNAi, GFP rotulagem podem ser acoplados com knockdown localizada de um determinado gene, o que permite determinar a resposta transcricional de uma população de células discretas para o silenciamento de genes específicos. Para validar esta abordagem, dissecamos áreas equivalentes do disco do posterior (rotulada pela expressão GFP), eo compartimento (sem identificação) anterior ao silenciamento regionais no compartimento de P de um gene de outra forma ubiquitously expressa. RNA foi extraído de microdissecção áreas silenciadas e unsilenced e perfil de expressão gênica comparativa determinado pelo quantitativo em tempo real, RT-PCR. Nós mostramos que este método pode ser aplicado de forma eficaz para transcriptômica precisa de subconjuntos de células dentro dos discos Drosophila imaginal. De fato, enquanto a preparação do disco massivo como fonte de RNA geralmente assume homogeneidade celular, é bem sabido que a expressão transcricional pode variar muito dentro dessas estruturas em conseqüência da informação posicional. Usando o sinal fluorescente GFP localizada a guia de corte a laser, mais precisas análises de transcrição pode ser realizada e rentável aplicado a diferentes aplicativos, incluindo perfis de transcrição de linhagens celulares distintas dentro de seu contexto nativo.

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

Microdissecção a laser foi aplicado para analisar perfil de expressão gênica em compartimentos específicos de Drosophila disco asa submetido a RNAi localizada<em&gt; In vivo</em&gt;. RNA extraído de áreas equivalentes de compartimentos silenciadas e unsilenced foi analisado por RT-PCR quantitativa para determinar perfis de expressão gênica comparativa no contexto da microecologia tecido nativo.

Microdissecção a laser Aplicada ao perfil de expressão gênica de subconjunto de células a partir do Drosophila Asa 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 visualizações.  University of Naples. Microdissecção a laser foi aplicado para analisar perfil de expressão gênica em compartimentos específicos de Drosophila disco asa submetido a RNAi localizada In vivo. RNA extraído de áreas equivalentes de compartimentos silenciadas e unsilenced foi analisado por RT-PCR quantitativa para determinar perfis de expressão gênica comparativa no contexto da microecologia tecido nativo.

Vídeo: 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.

Laser Microdissecção Captura de tecidos 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 ...

Biologia

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.

Elaboração de culturas de células primárias hematopoéticas de medula óssea de camundongos para Eletroporação

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.

Usando o Sistema de Eletroporação Gene Pulser MXcell para transfectar pilhas com Alta Eficiência

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.

Usando um contador de células automatizadas para simplificar Estudos da Expressão Gênica: Knockdown siRNA de Expressão IL-4 Gene Dependente em células 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.

Seções incluído em parafina e congelados de Drosophila Músculos Adulto

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.

Na reconstituição in vitro da atividade T. Telomerase 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.

Isolamento à base de Afinidade de Tagged Núcleos de<em&gt; Drosophila</em&gt; Tecidos para Análise de Expressão Gênica

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.

Análise da Global RNA Síntese no nível da célula única seguinte hipóxia

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 identificar Ubiquitin e ubiquitina-como reguladores do sistema de caminhos biológicos em células de mamíferos cultivadas

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.