Vorremmo ringraziare il Dott. Michael Baker per il suo aiuto nella generazione di costrutti per gene knock-in e per la prestazione del time-lapse film di crescere proiezioni sensoriali. Vorremmo anche ringraziare Virginia Vandelinder di assistenza tecnica con gli esperimenti della pistola pneumatica.

Particelle possono essere rivestiti con diversi reagenti, come le molecole dsRNA, plasmidi DNA o coloranti. Il tipo e il diametro delle particelle sono scelti in base al reagente e la profondità delle cellule bersaglio nei tessuti: le particelle più grandi penetrare ulteriormente, ma può causare qualche danno al tessuto. 1. Preparazione di particelle rivestito dsRNA <ol><li> Posto al 100% isopropanolo sul ghiaccio. </li><li> Risospendere 5 mg di particelle d&#39;oro (S1600ri, Seashell Technology, LLC; diametro medio di 1,6 micron) in 100 ml di tampone vincolanti. Quando la soluzione è pronta, passare alla fase successiva. </li><li> Diluire la soluzione di particelle aggiungendo 100 ml di buffer di legame, mescolare brevemente nel vortex e sonicare per 1-2 minuti per evitare grumi. </li><li> Aggiungi 5-10μg di dsRNA / siRNA (dsRNAs / siRNA deve essere sciolto in acqua ad una concentrazione di circa 1μg/μl). </li><li> Vortex e lasciare a temperatura ambiente per 2 minuti. Questi risultati intervallo di concentrazione e dal tempo in siRNA satura di particelle d&#39;oro. </li><li> Pellet il siRNA / oro complessi delle particelle mediante centrifugazione a ~ 2.500 giri in una centrifuga da banco per ~ 15 sec. </li><li> Rimuovere il surnatante e delicatamente aggiungere 750 ml di isopropanolo freddo con un&#39;interruzione minima del pellet. Spin brevemente e rimuovere il surnatante. </li><li> Risospendere le particelle d&#39;oro in 100 l freddo isopropanolo al 100%, poi sonicare brevemente in un bagno sonicatore (2-3 impulsi) per rompere agglomerati di particelle. Ultimo, versare le particelle in sospensione su un vetrino e lasciare asciugare a temperatura ambiente. </li></ol> 2. Preparare il setup sperimentale per un test specifico <ol><li> Ogni arma ha un diverso diametro di apertura degli ugelli, che vanno da un minimo di 50 micron fino a ~ ~ 3 mm. Le specifiche da utilizzare per la pistola è quindi scelto a seconda della zona di tessuto su cui agire. </li><li> Lui la pressione viene regolata per la profondità nel tessuto delle cellule bersaglio, in genere fino a ~ 100μm (alte pressioni in grado di fornire le particelle più profonda). La distanza tra l&#39;embrione e la punta dell&#39;ugello influisce anche sulla profondità di penetrazione, come momento d&#39;oro particelle libere in aria. </li></ol> 3. Caricamento della pistola con la pre-particelle rivestite Le particelle devono essere caricate come una polvere secca nelle linee di particelle tubi Tygon iniezione collegati al collettore. Questo tubo, che deve essere conservato in un ambiente asciutto a 4 ° C, quando non in uso, possono essere riutilizzati in ulteriori esperimenti con gli stessi reagenti. Il nostro apparato sperimentale in grado di fornire fino a due diversi reagenti per analisi attraverso porte separate nel collettore. <ol><li> Raschiare le particelle essiccate ricoperto dal vetro diapositive utilizzando il bordo di un vetrino di vetro o una lametta prima di caricarli nella linea di tubi Tygon. Questo tubo Tygon saranno specifiche solo per questo reagente per evitare contaminazioni incrociate. </li><li> Come si caricano le particelle, piegare il tubo a forma di U vicino al connettore, per mantenere le particelle di diffondersi lungo il tubo intero e concentrati vicino al connettore. Raccogliere le particelle con una piccola spatola o un pezzo di carta piegato pesano, e caricare circa la metà di loro nel tubo per ogni serie di embrioni. Toccare il tubo durante la fase di carico di diffondere le particelle in modo uniforme. </li></ol> 4. Preparazione degli animali Prima e dopo la consegna delle particelle d&#39;oro, gli embrioni vengono conservati in acqua sterile laghetto artificiale a temperatura ambiente. <ol><li> Mettere gli embrioni, lato ventrale in su, in un solco scavato in un appartamento, 5mm di spessore pezzo di gomma siliconica (Sylgard 184, Dow Corning, Midland, MI) e anestetizzare in una soluzione di 8% di etanolo in acqua sterile laghetto artificiale ( più giovani embrioni in fasi E6-E8 può rimanere in acqua laghetto artificiale senza etanolo tutto l&#39;esperimento). </li><li> Immediatamente prima di sparare le particelle, abbassare il livello bagno per scoprire la superficie ventrale degli embrioni, eliminando una parte della soluzione. Posizionare un pezzo di carta velina con un piccolo foro tagliati a metà su di esso per stabilizzare e mantenere la sua superficie si secchi. </li></ol> 5. Consegna di particelle rivestite in tessuto bersaglio Un carico di particelle in genere può essere utilizzato per un massimo di dieci scatti, con un solo colpo di solito consegna dell&#39;ordine di poche centinaia di particelle. <ol><li> Genera un colpo aprendo una delle valvole di 0,3 s, causando l&#39;iniezione di un bolo di particelle dalla linea di tubazione corrispondente nella pistola. </li><li> Toccare il tubo dolcemente tra gli scatti per rimuovere le particelle dal muro tubi e quindi facilitare la loro iniezione nel flusso di lui la pistola. </li><li> Per coprire l&#39;area di tessuto di interesse, scatti multipli possono essere richiesti. </li><li> Dopo la consegna delle particelle, posto che gli embrioni di nuovo in acqua laghetto artificiale, preferibilmente un embrione per bene in un multi-pozzetti. </li></ol> 6. Immunocolorazione e l&#39;etichettatura dei neuroni Mantenere gli embrioni a temperatura ambiente e al buio per 1 a 3 giorni dopo la consegna delle particelle, fino RNAi (o espressione ectopica) si ottiene. Abbiamo poi eseguire una delle seguenti procedure sui campioni sperimentali e controlli: <ol><li> Ibridazione in situ di test per la presenza o l&#39;assenza di mRNA netrina. Digossigenina marcato sanguisuga riboprobes netrina sono ibridate agli embrioni sanguisuga intero. </li><li> Immunocitochimica al test per le proteine ​​netrina e di altre proteine ​​che sono unici al tessuto neuronale (come anti tubulina acetilata). La procedura viene eseguita anche su tutto il montaggio degli embrioni. </li><li> Microiniezioni di coloranti lipofili (DII e Dio) in singoli neuroni, per la tintura riempie di etichettare i pergolati completa dei neuroni di interesse (neuroni pressione mechanosensory nel nostro caso) in segmenti trattate ei controlli. Questo test viene eseguito su embrioni vivi (intatti o sezionato). </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>

In questa dimostrazione, abbiamo usato la pistola pneumatica capillare per fornire particelle d&#39;oro all&#39;interno delle cellule localizzate in piccoli volumi di un embrione vivo sanguisuga per knock-in e knock-down geni. La regione del bersaglio in genere aveva un diametro di circa 150 micron e le particelle sono stati trovati circa 15 micron intorno alla profondità di penetrazione media, che è stato regolabile tra 0 e 50 micron. Quando le particelle sono stati rivestiti con molecole di dsRNA del netrina fattore guida degli assoni, ...

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<td>gold particles</td>
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<td>Seashell Technology, LLC</td>
<td>S1600ri</td>
<td>&nbsp;</td>
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<td>binding buffer</td>
<td>Reagent</td>
<td>Seashell Technology, LLC</td>
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<td>silicone rubber </td>
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localized rnai and ectopic gene expression in the medicinal leech

In questo video, che mostrano l&#39;uso di una pistola pneumatica capillare per la consegna accurata biolistic dei reagenti nel tessuto vivo. Usiamo la procedura per i modelli di espressione genica turbare in segmenti selezionati di embrioni sanguisuga, lasciando i segmenti non trattati come controlli interni. La pistola pneumatica capillare può essere utilizzato per raggiungere gli strati interni di cellule nelle fasi precoci dello sviluppo senza aprire il campione. Come metodo per l&#39;introduzione di sostanze localizzato nei tessuti viventi, la consegna biolistic con la pistola ha diversi vantaggi: è veloce, senza contatto e non distruttiva. Inoltre, una pistola singolo capillare può essere utilizzato per la consegna indipendente di sostanze diverse. La regione di consegna può avere dimensioni laterali di circa 50-150 micron e si estende su circa 15 micron intorno alla profondità di penetrazione media, che è regolabile tra 0 e 50 micron. Questa consegna è il vantaggio di essere in grado di indirizzare un numero limitato di celle in una posizione intermedia tra selezionato singola cella abbattere da microiniezione e knockdown sistemico attraverso iniezioni extracellulare o mediante approcci genetici. Per abbattere o bussare nell&#39;espressione del netrina molecola guida degli assoni, che è naturalmente espresso da alcuni neuroni centrali e nella parete ventrale del corpo, ma non il dominio dorsale, forniamo le molecole di dsRNA o plasmide-DNA nella parete del corpo e gangli centrali. Questa procedura prevede le seguenti fasi: (i) la preparazione del setup sperimentale per un test specifico (la regolazione della pressione di accelerazione), (ii) il rivestimento di particelle con le molecole di dsRNA o DNA, (iii) le particelle di carico rivestito nella pistola, fino a due reagenti in un saggio, (iv) preparare gli animali per la consegna delle particelle, (v) la consegna di particelle rivestite in tessuto bersaglio (parete del corpo o gangli), e (vi) il trattamento degli embrioni (immunostaining, immunoistochimica e neuronali etichettatura) per visualizzare i risultati, di solito 2 o 3 giorni dopo il parto. Quando le particelle sono stati rivestiti con netrina dsRNA, hanno causato ben visibili knock-down di espressione netrina che si è verificato solo nelle cellule contenenti particelle (in genere, 1-2 particelle per cella). Particelle rivestite con un plasmide fluorescenza EGFP codifica indotta in cellule neuronali quando si fermarono nel loro nucleo.

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

In questo video vi mostriamo una procedura per una consegna precisa biolistic dei reagenti nel tessuto vivo con una pistola romanzo gene in miniatura. Stiamo abbattendo l&#39;espressione della molecola netrina guida degli assoni negli embrioni sanguisuga fornendo molecole di dsRNA nella parete ventrale del corpo e dei gangli di singoli segmenti.

Localizzato RNAi e di espressione genica ectopica nel Leech medicinali

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 Views.  University of California San Diego - UCSD. In questo video vi mostriamo una procedura per una consegna precisa biolistic dei reagenti nel tessuto vivo con una pistola romanzo gene in miniatura. Stiamo abbattendo l'espressione della molecola netrina guida degli assoni negli embrioni sanguisuga fornendo molecole di dsRNA nella parete ventrale del corpo e dei gangli di singoli segmenti.

Video: 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.

La preparazione di colture primarie di cellule emopoietiche dal midollo osseo murino per elettroporazione

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

Ricerca

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 Elettrofisiologia

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.

Utilizzando il sistema di elettroporazione Gene Pulser MXcell per trasfettare cellule primarie con alta efficienza

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.

Robotica e Image Analysis dinamica per gli Studi di espressione genica nei tessuti vegetali

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.

L&#39;utilizzo di un contatore automatico cella per semplificare gli studi sull&#39;espressione genica: Knockdown siRNA di IL-4 genica dipendente nelle celle 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.

Sezioni incluse in paraffina e surgelati di Drosophila Adulti Muscoli

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.

Ricostituzione in vitro di Active T. castaneum telomerasi

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 mediata Knockdown Gene e Transgenesi dalla microiniezione nel nematode 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.

Analisi globale di RNA Sintesi a livello di singola cellula seguente ipossia

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.