Gostaríamos de agradecer ao Dr. Michael Baker pela sua ajuda na geração de construções para gene knock-in e por fornecer os filmes de lapso de tempo de crescer projeções sensoriais. Gostaríamos também de agradecer a Virginia Vandelinder de assistência técnica com os experimentos pistola pneumática.

Partículas podem ser revestidos com reagentes diferentes, tais como moléculas de dsRNA, plasmídeos DNA ou corantes. O tipo eo diâmetro das partículas são escolhidos de acordo com o reagente e da profundidade das células-alvo no tecido: partículas maiores penetrar mais, mas pode causar algum dano ao tecido. 1. Preparação de partículas dsRNA revestido <ol><li> Coloque 100% isopropanol no gelo. </li><li> Ressuspender 5 mg partículas de ouro (S1600ri, Seashell Technology, LLC; diâmetro médio 1,6 mm) em 100 ml de tampão de ligação. Quando a solução está pronto, continue para a próxima etapa. </li><li> Diluir a solução de partículas, adicionando 100 ul de tampão de ligação, brevemente vórtice e sonicate por 1-2 min, para evitar aglomeração. </li><li> Adicionar 5-10μg de dsRNA / siRNA (dsRNAs / siRNA deve ser dissolvido em água a uma concentração de ~ 1μg/μl). </li><li> Vortex e deixe em temperatura ambiente por 2 min. Estes resultados intervalo de concentração e tempo em siRNA saturada partículas de ouro. </li><li> Pellet os complexos siRNA / ouro de partículas por centrifugação a 2.500 rpm ~ em uma centrífuga de bancada para ~ 15 seg. </li><li> Remover o sobrenadante e adicionar 750 ml gentilmente isopropanol frio com mínima interrupção do sedimento. Girar rapidamente e remover o sobrenadante. </li><li> Ressuspender as partículas de ouro em 100 l frio isopropanol 100%, então sonicate brevemente em um sonicador banho (2-3 pulsos) para acabar com aglomerados de partículas. Passado, despeje a partículas em suspensão sobre uma lâmina de vidro e deixar secar à temperatura ambiente. </li></ol> 2. Preparando a instalação experimental para um teste específico <ol><li> Cada arma tem um diâmetro de abertura do bocal diferentes, variando de um mínimo de 50 ìm ~ até ~ 3mm. Específico a ser utilizado para a arma é, portanto, escolhidos de acordo com a área de tecido a ser alvo. </li><li> A pressão Ele é ajustado para a profundidade no tecido das células-alvo, geralmente até ~ 100μm (pressões mais elevadas podem entregar as partículas mais profundo). A distância entre o embrião ea ponta do bico também afeta a profundidade de penetração, como momento de ouro partículas soltas no ar. </li></ol> 3. Carregando a arma com as partículas pré-revestidas Partículas devem ser carregados como um pó seco no tubo tygon linhas de injeção de partículas ligado ao colector. Essa tubulação, que deve ser mantido em um ambiente seco, a 4 ° C, quando não em uso, pode ser re-utilizados em experimentos adicionais com os mesmos reagentes. Nossa configuração experimental pode fornecer até dois reagentes diferentes por ensaio através de portas separadas no coletor. <ol><li> Raspe as partículas secas revestido da lâmina pela beira de um lamínula de vidro ou uma lâmina de barbear antes de colocá-los na linha de tubos tygon. Este tubo tygon serão específicas apenas para este reagente para evitar a contaminação cruzada. </li><li> Como você carregar as partículas, dobre o tubo em forma de U perto do conector, para manter as partículas se espalhe ao longo de toda a tubulação e concentrada perto do conector. Pegue as partículas com uma pequena espátula ou um pedaço de papel dobrado pesar e carregar cerca de metade deles no tubo para cada série de embriões. Toque no tubo durante a etapa de carregamento para espalhar as partículas uniformemente. </li></ol> 4. Preparação dos animais Antes e após a entrega das partículas de ouro, os embriões são mantidos em água da lagoa artificial estéril à temperatura ambiente. <ol><li> Coloque os embriões, o lado ventral para cima, em um sulco esculpido em um apartamento, um pedaço cinco milímetros de espessura de borracha de silicone (Sylgard 184, Dow Corning, Midland, MI) e anestesiar-los em uma solução de etanol a 8% em água estéril lagoa artificial ( embriões em estágios mais jovens E6-E8 pode permanecer na água da lagoa artificial, sem o etanol durante todo o experimento). </li><li> Imediatamente antes de disparar as partículas, menor o nível do banho de descobrir a superfície ventral dos embriões através da remoção de parte da solução. Coloque um pedaço de papel de seda com um pequeno buraco cortado no meio em cima dela para estabilizá-lo e para manter sua superfície de secagem. </li></ol> 5. Entrega de partículas revestidas em tecido-alvo Uma carga de partículas geralmente pode ser usado por até dez tiros, com um único tiro normalmente cumprindo a ordem de algumas centenas de partículas. <ol><li> Gerar um tiro, abrindo uma das válvulas para 0,3 s, fazendo com que a injeção de um bolus de partículas a partir da linha correspondente na tubulação de arma. </li><li> Toque suavemente o tubo entre os tiros para desalojar as partículas da parede tubulação e, assim, facilitar a sua injecção na corrente Ele da arma. </li><li> Para cobrir a área de tecido de interesse, vários tiros podem ser necessárias. </li><li> Após a entrega das partículas, coloque os embriões de volta à água da lagoa artificial, de preferência um embrião por bem em uma placa multi-bem. </li></ol> 6. Imunocoloração e rotulagem neuronal Manter os embriões em temperatura ambiente e no escuro por 1 a 3 dias após a entrega de partículas, até RNAi (ou expressão ectópica) seja alcançado. Em seguida, realizar um dos procedimentos a seguir sobre as amostras experimentais e controles: <ol><li> Hibridização in situ de ensaio para a presença ou ausência de mRNA netrin. Marcada com digoxigenina riboprobes leech netrin são cruzados com embriões leech todo. </li><li> Imunocitoquímica para ensaio para netrin proteínas e outras proteínas que são únicas para o tecido neuronal (como anti tubulina acetilada). O procedimento é realizado também em toda montagem embriões. </li><li> Microinjeções de corantes lipofílicos (DII e Dio) em neurônios individuais, para tingir-enche de rotular os mandris completa dos neurônios de interesse (neurônios pressão mecanosensorial no nosso caso) nos segmentos de controle e tratados. Este ensaio é realizado em embriões vivos (intactos ou dissecado). </li></ol>

<ol>
	<li>Baker, M. W., Macagno, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi+of+the+receptor+tyrosine+phosphatase+HmLAR2+in+a+single+cell+of+an+intact+leech+embryo+leads+to+growth-cone+collapse.">RNAi of the receptor tyrosine phosphatase HmLAR2 in a single cell of an intact leech embryo leads to growth-cone collapse.</a> Current Biology. 10, 1071-1074 (2000).</li><li>Baker, M. W., Macagno, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Hirudo+medicinalis+DNA+Promoters+for+Targeted+Gene+Expression.">Characterization of Hirudo medicinalis DNA Promoters for Targeted Gene Expression.</a> Journal of Neuroscience Methods. 156, 145-153 (2006).</li><li>Gan, W. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+expression+of+a+leech+netrin+suggests+roles+in+the+formation+of+longitudinal+nerve+tracts+and+in+regional+innervation+of+peripheral+targets.">Cellular expression of a leech netrin suggests roles in the formation of longitudinal nerve tracts and in regional innervation of peripheral targets.</a> Journal of Neurobiology. 40, 103-115 (1999).</li><li>Juven-Gershon, T., Cheng, S., Kadonaga, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+of+a+super+core+promoter+that+enhances+gene+expression.">Rational design of a super core promoter that enhances gene expression.</a> Nature Methods. 3, 917-922 (2006).</li><li>Rinberg, D., Simonnet, C., Groisman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pneumatic+capillary+gun+for+ballistic+delivery+of+microparticles.">Pneumatic capillary gun for ballistic delivery of microparticles.</a> Applied Physics Letters. 87, 014103 (2005).</li><li>Shefi, O., Simonnet, C., Baker, M. W., Glass, J. R., Macagno, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtargeted+gene+silencing+and+ectopic+expression+in+live+embryos+using+biolistic+delivery+with+a+pneumatic+capillary+gun.">Microtargeted gene silencing and ectopic expression in live embryos using biolistic delivery with a pneumatic capillary gun.</a> Journal of Neuroscience. 26, 6119-6123 (2006).</li></ol>

Nesta demonstração, utilizamos a pistola pneumática capilar para entregar partículas de ouro em células localizadas no volume pequeno de um embrião vivo para sanguessuga knock-in e knock-down genes. A região-alvo, geralmente tinha um diâmetro de ~ 150 mm e partículas foram encontrados ~ 15 mm em torno da profundidade de penetração média, que era ajustável entre 0 e 50 mm. Quando as partículas foram revestidos com moléculas de dsRNA do netrin fator de orientação axônio, eles causaram claramente visível knock-down de netrin...

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<td>gold particles</td>
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<td>Seashell Technology, LLC</td>
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localized rnai and ectopic gene expression in the medicinal leech

Neste vídeo, mostramos o uso de uma pistola pneumática capilar para a entrega precisa biobalística de reagentes em tecido vivo. Nós usamos o procedimento de padrões de expressão gênica perturb em segmentos selecionados de embriões sanguessuga, deixando os segmentos não tratados como controles internos. A pistola pneumática capilar pode ser utilizada para atingir camadas internas de células em estágios iniciais de desenvolvimento sem abrir o espécime. Como um método para a introdução de substâncias localizadas em tecidos vivos, a entrega biobalística com a arma tem várias vantagens: é rápido, sem contato e não-destrutivos. Além disso, uma arma única capilar pode ser utilizado para a entrega independente de substâncias diferentes. A região de entrega pode ter dimensões laterais de ~ 50-150 mm e se estende por ~ 15 mm em torno da profundidade de penetração média, que é ajustável entre 0 e 50 mm. Esta entrega tem a vantagem de ser capaz de atingir um número limitado de células em um local selecionado intermediário entre célula única derrubar por microinjeção e knockdown sistêmica através de injeções extracelular ou por meio de abordagens genéticas. Para derrubar ou bater na expressão da molécula de orientação netrin axônio, que é naturalmente expresso por alguns neurônios centrais e na parede ventral do corpo, mas não o domínio do dorsal, nós entregamos moléculas de dsRNA ou DNA-plasmídeo na parede do corpo e gânglios central. Este procedimento inclui as seguintes etapas: (i) preparação da montagem experimental para um teste específico (ajustando a pressão de aceleração), (ii) revestimento das partículas com as moléculas de dsRNA ou DNA, (iii) o carregamento das partículas revestido na arma, até dois reagentes em um ensaio, (iv) preparar os animais para a entrega de partículas, (v) a entrega de partículas revestidas no tecido alvo (parede do corpo ou gânglios), e (vi) o tratamento dos embriões (imunohistoquímica de positividade, e neuronal rotulagem) para visualizar os resultados, geralmente 2 a 3 dias após o parto. Quando as partículas foram revestidas com netrin dsRNA, que causaram claramente visível knock-down de netrin expressão que só ocorreu em células contendo partículas (geralmente, 1-2 partículas por célula). Partículas revestidas com uma codificação de fluorescência EGFP plasmídeo induzida em células neuronais quando pararam em seus núcleos.

Watch this Scientific Journal Video about Localized RNAi and Ectopic Gene Expression in the Medicinal Leech at JoVE.com

Localized RNAi and Ectopic Gene Expression in the Medicinal Leech

Neste vídeo, mostramos um procedimento para uma entrega precisa biobalística de reagentes em tecido vivo com uma arma gene romance em miniatura. Estamos derrubando a expressão da molécula de orientação netrin axônio em embriões de sanguessuga, fornecendo moléculas de dsRNA na parede ventral do corpo e gânglios dos segmentos individuais.

Localizada RNAi e Expressão Gênica ectópica na sanguessuga medicinal

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to ...

localized-rnai-and-ectopic-gene-expression-in-the-medicinal-leech

Research

JoVE Journal

Biology

9.5K visualizações.  University of California San Diego - UCSD. Neste vídeo, mostramos um procedimento para uma entrega precisa biobalística de reagentes em tecido vivo com uma arma gene romance em miniatura. Estamos derrubando a expressão da molécula de orientação netrin axônio em embriões de sanguessuga, fornecendo moléculas de dsRNA na parede ventral do corpo e gânglios dos segmentos individuais.

Vídeo: Localized RNAi and Ectopic Gene Expression in the Medicinal Leech

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 ...

Biologia

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

Spinal Cord Eletrofisiologia

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

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

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

Robótica e Análise de Imagem Dinâmica de Estudos de Expressão Gênica em Tecidos Vegetais

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

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 ...

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans3, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis4 and Pristionchus pacificus5. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior6-7, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers5. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis4 and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms8. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F1 progeny. Unlike C. elegans, but like most other nematodes, P. pacificus PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4 promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92) mutant in a visually informative protocol.

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

In model organisms, transgenesis can manipulate gene functions while RNAi can knockdown specific mRNA transcripts 1-2. This protocol aims to illustrate the techniques needed to introduce stably transmitted DNA and transient double stranded RNA into the necromenic nematode Pristionchus pacificus for studies in evolutionary, developmental, and behavioral biology.

RNAi Mediated Gene Knockdown e Transgénese por microinjeção no Nematóides Necromenic Pristionchus pacificus</em

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression ...

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 ...

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.