Wir möchten Dr. Michael Baker für seine Hilfe danken Erzeugung Konstrukte für die Gen-knock-in und für die Bereitstellung der Zeitraffer-Filme wachsender sensorischen Projektionen. Wir möchten auch Virginia Vandelinder für technische Hilfe mit dem Luftgewehr Experimente danken.

Partikel können mit verschiedenen Reagenzien, wie dsRNA Moleküle, DNA-Plasmiden oder Farbstoffe aufgebracht werden. Die Art und Durchmesser der Teilchen nach dem Reagenz und die Tiefe der Zielzellen in das Gewebe gewählt: größere Partikel dringen weiter, können aber eine Schädigung des Gewebes führen. 1. Vorbereitung der dsRNA beschichteten Partikel <ol><li> Legen Sie 100% Isopropanol auf Eis. </li><li> Resuspendieren 5 mg Goldpartikel (S1600ri, Seashell Technology, LLC, mittleren Durchmesser 1,6 um) in 100 ml Bindungspuffer. Als Lösung bereit ist, mit dem nächsten Schritt fortfahren. </li><li> Verdünnen Sie die Partikel-Lösung durch Zugabe von 100 ul Bindungspuffer, kurz vortexen und beschallen für 1-2 min zu vermeiden, zu verklumpen. </li><li> Add 5-10 ug von dsRNA / siRNA (dsRNAs / siRNA sollte in Wasser auf eine Konzentration von ~ 1μg/μl aufgelöst werden). </li><li> Vortex und lassen bei Raumtemperatur für 2 min. Diese Konzentration und Zeitintervall Ergebnis in siRNA gesättigten Goldpartikel. </li><li> Pellet der siRNA / Goldpartikel Komplexe durch Zentrifugation bei ~ 2.500 upm in einer Tischzentrifuge für ~ 15 sek. </li><li> Entfernen Sie den Überstand und sanft add 750 ml kaltem Isopropanol mit minimaler Unterbrechung des Pellets. Spin kurz und entfernen Sie den Überstand. </li><li> Resuspendieren Goldpartikel in 100 ul kaltem 100% Isopropanol, dann kurz beschallen in einem Ultraschallbad (2-3 Pulse) zu brechen Partikelagglomerate. Last, gießen Sie die Schwebeteilchen auf einen Objektträger aus Glas und lassen Sie sie bei Raumtemperatur trocknen. </li></ol> 2. Vorbereiten des Versuchsaufbaus für einen bestimmten Test <ol><li> Jede Waffe hat eine andere Düsenöffnung Durchmesser reicht von einem Minimum von ~ 50 um bis zu ~ 3mm. Die spezifische, für die Waffe verwendet werden soll daher nach dem Gebiet des Tissue gezielt gewählt. </li><li> Die He-Druck ist für die Tiefe in das Gewebe der Zielzellen angepasst, in der Regel bis zu ~ 100 um (höhere Drücke können die Teilchen tiefer zu liefern). Der Abstand zwischen dem Embryo und die Spitze der Düse wirkt sich auch auf die Eindringtiefe, wie Goldpartikel lockeren Schwung in der Luft. </li></ol> 3. Laden der Waffe mit der Pre-beschichtete Partikel Partikel sollten als trockenes Pulver in die Tygon-Schlauch Partikelinjektion Linien verbunden, um die vielfältigen geladen werden. Diese Schläuche, die in einer trockenen Atmosphäre bei 4 gehalten werden sollte ° C, wenn nicht in Gebrauch ist, kann in weiteren Experimenten mit dem gleichen Reagenzien wieder verwendet werden. Unser Versuchsaufbau kann bis zu zwei verschiedenen Reagenzien pro Test durch separate Ports im Verteiler. <ol><li> Kratzen Sie die getrockneten beschichteten Partikel aus dem Glasträger mit der Kante eines Deckglas oder einer Rasierklinge vor dem Einlegen in die Tygon-Schlauch Linie. Dieser Tygon-Schlauch wird nur für diesen Reagenz, um eine Kreuzkontamination zu vermeiden. </li><li> Wie Sie die Partikel zu laden, beugen Sie den Schlauch in eine U-Form in der Nähe der Stecker, um die Partikel Ausbreitung entlang der gesamten Schlauch und konzentrierte sich in der Nähe der Anschluss zu halten. Pick-up die Partikel mit einem kleinen Spatel oder einem Stück gefaltet wiegen Papier, und legen etwa die Hälfte davon in den Schlauch für jede Serie von Embryonen. Tippen Sie auf den Schlauch während der Beladung Schritt, um die Partikel gleichmäßig verteilt. </li></ol> 4. Tierische Vorbereitung Vor und nach der Goldpartikel Lieferung, werden die Embryonen in sterile künstlichen Teich Wasser bei Raumtemperatur aufbewahrt. <ol><li> Legen Sie die Embryonen, Bauchseite nach oben in eine Nut in eine flache, 5mm-dickes Stück aus Silikon-Kautschuk (Sylgard 184, Dow Corning, Midland, MI) geschnitzt und betäuben sie in eine Lösung von 8% Ethanol in sterile künstlichen Teich Wasser ( jüngeren Embryonen in den Stadien E6-E8 kann in künstlichen Teich Wasser ohne Ethanol während des gesamten Experiments bleiben). </li><li> Unmittelbar vor der Aufnahme der Partikel niedriger der Badspiegel der ventralen Oberfläche des Embryos, indem ein Teil der Lösung aufzudecken. Legen Sie ein Stück Seidenpapier mit einem kleinen Loch in der Mitte oben drauf geschnitten, um es zu stabilisieren und die Oberfläche vor dem Austrocknen bewahren. </li></ol> 5. Lieferung von beschichteten Partikel in das Zielgewebe Eine Belastung der Partikel können in der Regel für bis zu zehn Aufnahmen verwendet werden, mit einem einzigen Schuss in der Regel liefert der Größenordnung von ein paar hundert Partikel. <ol><li> Generieren Sie einen Schuss durch das Öffnen eines der Ventile für 0,3 s, so dass die Injektion eines Bolus von Partikeln aus der entsprechenden Rohrleitung in die Pistole. </li><li> Tippen Sie den Schlauch vorsichtig zwischen den Aufnahmen, um Partikel aus dem Schlauch Wand lösen und erleichtern somit die Injektion in die He-Strom der Waffe. </li><li> Zur Abdeckung der Fläche von Gewebe von Interesse, können mehrere Schüsse erforderlich sein. </li><li> Nach der Partikel Lieferung, statt die Embryonen wieder in künstlichen Teich Wasser, vorzugsweise ein Embryo pro Well in einer Multi-Well-Platte. </li></ol> 6. ImmunoFärbung und neuronalen Kennzeichnung Halten Sie die Embryonen bei Raumtemperatur und in Dunkelheit für 1 bis 3 Tage nach der Partikel Lieferung bis RNAi (oder ektopische Expression) ist erreicht. Wir führen Sie dann eine der folgenden Verfahren auf der experimentellen Proben und Kontrollen: <ol><li> In-situ-Hybridisierung zum Testen auf das Vorhandensein oder Fehlen von Netrin mRNA. Digoxigenin-markierten Blutegel Netrin Ribosonden sind ganze Blutegel Embryonen hybridisiert. </li><li> Immunzytochemie zum Testen auf Netrin und anderen Proteinen, die einzigartig für neuronales Gewebe (wie z. B. Anti acetyliertes Tubulin) sind. Das Verfahren ist auch auf ganze-mount Embryonen durchgeführt. </li><li> Mikroinjektionen lipophiler Farbstoffe (DII und DIO) in einzelnen Neuronen, für Farbstoff-füllt den kompletten Lauben der Neuronen von Interesse (mechanosensorischen Druck Neuronen in unserem Fall) in Behandlungs-und Kontrollgruppe Segmente Etikett. Dieser Assay basiert auf Live-Embryonen (intakt oder seziert) durchgeführt. </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 dieser Demonstration, verwendeten wir die pneumatische Kapillare Pistole an Goldpartikel in Zellen liefern in kleinen lokalisierten Volumen von einer Live-leech Embryo bis zum Knock-in und knock-down-Gene. Die Zielregion der Regel einen Durchmesser von ~ 150 um und Partikel gefunden wurden ~ 15 um rund um die mittlere Eindringtiefe, die zwischen 0 und 50 um. Wenn die Teilchen mit dsRNA Moleküle des Axons Führung Faktor Netrin beschichtet waren, verursachten sie deutlich sichtbar knock-down von Netrin Ausdruck, der nur in Zellen mit Par...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>gold particles</td>
<td>Other</td>
<td>Seashell Technology, LLC</td>
<td>S1600ri</td>
<td>&nbsp;</td>
<tr>
<td>binding buffer</td>
<td>Reagent</td>
<td>Seashell Technology, LLC</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>silicone rubber </td>
<td>Reagent</td>
<td>Dow Corning</td>
<td>Sylgard 184</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

localized rnai and ectopic gene expression in the medicinal leech

In diesem Video zeigen wir die Verwendung eines pneumatischen Kapillare Waffe für die genaue biolistischen Lieferung von Reagenzien in lebendem Gewebe. Wir nutzen das Verfahren zur stören Genexpressionsmuster in ausgewählten Segmenten des Blutegel Embryonen, so dass die unbehandelten Segmenten als interne Kontrollen. Die pneumatische Kapillare Waffe kann zur inneren Schichten von Zellen in einem frühen Stadium der Entwicklung erreicht, ohne Öffnung der Probe sein. Als Verfahren zur lokalisierten Einbringen von Stoffen in lebenden Geweben, die biolistischen Lieferung mit der Pistole mehrere Vorteile: Sie ist schnell, berührungslos und zerstörungsfrei. Darüber hinaus kann eine einzelne Kapillare Pistole für unabhängige Lieferung von verschiedenen Substanzen verwendet werden. Die Lieferung einer Region kann auch lateralen Abmessungen von ca. 50-150 um und erstreckt sich über ca. 15 um rund um die mittlere Eindringtiefe, die zwischen 0 und 50 um liegt. Diese Auslieferung hat den Vorteil, dass in der Lage, eine begrenzte Anzahl von Zellen in einem ausgewählten Ort der Mitte zwischen einzelnen Zelle knock down durch Mikroinjektion und systemische Knockdown durch extrazelluläre Injektionen oder durch genetische Ansätze Ziel. Für umzuwerfen oder Klopfen in der Expression des Axons Führung Molekül Netrin, die natürlich von einigen zentralen Neuronen und in der ventralen Körperwand zum Ausdruck kommt, nicht aber die dorsale Domäne, liefern wir Moleküle dsRNA oder Plasmid-DNA in den Körper Wand-und zentralen Ganglien. Dieses Verfahren umfasst folgende Schritte: (i) Vorbereitung der Versuchsaufbau für einen spezifischen Assay (Anpassung der Beschleunigung Druck), (ii) die Beschichtung der Partikel mit Molekülen der dsRNA oder DNA, (iii) das Laden der beschichteten Partikel in die Pistole, bis zu zwei Reagenzien in einem Test, (iv) die Vorbereitung der Tiere für die Partikel-Lieferung, (v) Lieferung von beschichteten Partikel in das Zielgewebe (Körper Wand oder Ganglien), und (vi) die Verarbeitung der Embryonen (Immunfärbung, Immunhistochemie und neuronalen Kennzeichnung), um die Ergebnisse zu visualisieren, in der Regel 2 bis 3 Tage nach der Lieferung. Wenn die Teilchen mit Netrin dsRNA beschichtet waren, verursachten sie deutlich sichtbar knock-down von Netrin Ausdruck, der nur in Zellen mit Teilchen (in der Regel, 1-2 Partikel pro Zelle) aufgetreten ist. Partikel mit einem Plasmid kodiert EGFP Fluoreszenz in neuronalen Zellen, wenn sie in ihrem Kern nicht mehr beschichtet.

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 diesem Video zeigen wir ein Verfahren für eine genaue biolistischen Lieferung von Reagenzien in lebendes Gewebe mit einem neuartigen Mini-Gen-Kanone. Wir sind umzuwerfen der Ausdruck der Axon Führung Molekül Netrin in Blutegel Embryonen durch die Bereitstellung Moleküle dsRNA in der ventralen Körperwand und Ganglien der einzelnen Segmente.

Lokalisierte RNAi und ektopische Genexpression in der Blutegel

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 Aufrufe.  University of California San Diego - UCSD. In diesem Video zeigen wir ein Verfahren für eine genaue biolistischen Lieferung von Reagenzien in lebendes Gewebe mit einem neuartigen Mini-Gen-Kanone. Wir sind umzuwerfen der Ausdruck der Axon Führung Molekül Netrin in Blutegel Embryonen durch die Bereitstellung Moleküle dsRNA in der ventralen Körperwand und Ganglien der einzelnen Segmente.

Serie: 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.

Die Herstellung von primären hämatopoetischen Zellkulturen aus Knochenmark der Maus für die Elektroporation

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

Forschung

Biologie

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 Elektrophysiologie

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.

Mit dem Gene Pulser MXcell Elektroporation System zur Primary Cells mit hohem Wirkungsgrad zu transfizieren

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.

Robotik und Dynamic Image Analysis für Studies of Gene Expression in pflanzlichen Geweben

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.

Verwendung eines automatisierten Cell Counter zu Genexpressionsstudien Simplify: siRNA Knockdown von IL-4 abhängige Genexpression in Namalwalzellen

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.

Paraffin-Embedded-und Gefrierschnitte von Drosophila Adult Muskeln

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.

In vitro Rekonstitution der 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 ...

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-vermittelte Gene Knockdown und Transgenese durch Mikroinjektion in die Necromenic Nematode 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.

Analyse der globalen RNA-Synthese auf der Einzelzellebene folgende Hypoxie

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.