Diese Arbeit wurde von der Pflanze Genome Research Program von der National Science Foundation (NSF-PGRP-IOS-1339388) unterstützt.

1. Wachstum der Pflanzen
<ol><li>Säen von 5-10 Soja (Williams 82) in einem 13 cm-Topf im Gewächshaus unter Bedingungen der langen Tage (16 h Licht bei 1.500 µmol m-2 s-1) bei 25 ° C auf die benutzerdefinierte Erdmischung für Soja (1:1:1 Verhältnis von Erde, Perlite und Torpedo Sand).</li>
</ol>2. Vorbereitung von Plasmid DNA
<ol><li>Mit einem sterilen Pipettenspitze oder Zahnstocher, wählen Sie eine einzelne Kolonie oder gefrorenen Glycerin Lager von E. Coli tragen das Plasmid enthält das gen des Interesses und in 20 mL Luria-Bertani (LB) flüssigen Medium mit geeigneten Antibiotika in ein 50 mL Fläschchen zu impfen.</li>
	<li>Inkubieren Sie die Flasche bei 37 ° C über Nacht in einen Shaker geben. Sammeln Sie die Bakterien, indem die Federung bei 12.000 x g bei Raumtemperatur für 5 min Zentrifugieren und den Überstand verworfen. Extrahieren Sie und zu reinigen Sie das Plasmid des Herstellers Verfahren ein Plasmid Prep Kit.</li>
</ol>(3) Protoplasten Isolierung
<ol><li>Schneiden Sie neu erweiterten unifoliate Blätter von 10 - Tage alten Sojabohnen Keimlinge (Abbildung 1). Hinweis: Auswahl von Blättern in einer entsprechenden Entwicklungsstadium ist der Schlüssel zum Erfolg für Sojabohnen Protoplasten Vorbereitung. Verwenden Sie nur gerade erweitert Blätter in frühen Entwicklungsstadien. Als ältere Blätter werden Zellwände schwieriger um zu verdauen.</li>
	<li>Mit einer frischen Rasierklinge entfernen der Mittelrippe aus der unifoliate Blatt und dann schneiden Sie die Reste in 0,5-1 mm Streifen. Hinweis: Zwei unifoliate Blätter in 10 mL Enzymlösung verdaut geben ausreichende Protoplasten für mehr als 10 Transformationen.</li>
	<li>Das Blatt Streifen mit einer Zange, Transfer sofort und sanft in 10 mL frisch zubereitete Enzymlösung (Tabelle 1) in der 15 mL Tube. Vakuum Infiltrat Streifen das Blatt für 15 min bei Raumtemperatur.</li>
	<li>Inkubieren Sie die Blatt-Streifen in der Enzymlösung mit sanften Agitation (40 u/min) unter schlechten Lichtverhältnissen für 4-6 h bei Raumtemperatur. Stellen Sie sicher, dass das Enzymlösung Gelb-Grün dreht, da Protoplasten veröffentlicht werden. Überprüfen Sie die Enzym/Protoplasten Lösung unter dem Mikroskop (X10). Hinweis: Die veröffentlichten Protoplasten sind kugelförmige geprägt, während die unverdauten Zellen unregelmäßig oder ovale Form haben.</li>
	<li>Übertragen Sie die 10 mL der Lösung Enzym/Protoplasten in einem 50 mL-Tube vorsichtig gießen und 10 mL W5 Lösung (Tabelle 1) bei Raumtemperatur, die Verdauung zu stoppen. Invertieren Sie das Rohr sanft ein paar Mal. Füllen Sie vorsichtig die Enzym/Protoplasten Lösung auf eine saubere 75 µm-Nylon-Mesh platziert eine 50 mL-Tube, unverdaute Blatt Gewebe zu entfernen.</li>
	<li>Zentrifugieren Sie durchströmten Enzym/Protoplasten Lösung bei 100 X g in der 50 mL Tube für 1-2 min bei Raumtemperatur. Entfernen Sie vorsichtig den Überstand mit einer serologischen 10 mL-Pipette ohne störende Protoplasten Pellet.</li>
	<li>Die Protoplasten Aufschwemmen und zu einer Konzentration von 2 x 105 mL-1 in gekühlten W5-Lösung bei 4 ° C von Protoplasten zählen auf eine Hemacytometer unter dem Mikroskop (x10) verdünnen. Halten Sie die Protoplasten für 30 min auf Eis.</li>
	<li>Zentrifugieren Sie die Suspension 100 X g für 1-2 min bei Raumtemperatur und entfernen Sie vorsichtig die W5-Lösung mit einer 1-mL-Pipette ohne störende Protoplasten Pellet. Aufschwemmen der Protoplasten in MMG Lösung (Tabelle 1) in einer Konzentration von 2 x 105 mL-1 bei Raumtemperatur.</li>
</ol>4. Protoplasten transformation
<ol><li>Machen Sie 100 µL-Aliquots von Protoplasten (2 x 104 Protoplasten bei 2 x 105 mL-1) in 1,5 mL geringe Haftung Mikrozentrifugenröhrchen mit unbeschnittenen 200 µL Pipettenspitzen. Setzen Sie eine aliquote beiseite, um als negative Kontrolle dienen. Fügen Sie 10 µL Plasmid (10-20 µg) in jede der übrigen aliquoten von Protoplasten.</li>
	<li>Fügen Sie langsam 110 µL frisch zubereitete PEG-Lösung (Tabelle 1 hinzu) an der Innenwand der 1,5 mL Microcentrifuge Schlauch, und dann invertieren Sie sanft zu und drehen Sie das Rohr, bis die Lösung homogen wird.</li>
	<li>Inkubieren Sie die Verwandlung-Mischung bei Raumtemperatur für 15 min.</li>
	<li>Um die Transformation zu stoppen, der 1,5 mL Tube bei Raumtemperatur langsam 400 µL W5-Lösung hinzu und invertieren Sie das Rohr sanft zu, bis die Lösung homogen wird. Zentrifugieren Sie das Rohr bei 100 g für 1-2 min bei Raumtemperatur und verwerfen Sie den Überstand mit einer Pipette zu.</li>
	<li>Das Rohr 1 mL WI-Lösung (Tabelle 1) hinzu und Aufschwemmen von 1-bis 2-mal sanft pipettieren. Fügen Sie 1 mL 5 % (Vol/Vol) sterile Kälberserum in jede Vertiefung einer Gewebekultur 6-Well-Platte Beschichten der Oberfläche zu verhindern, dass die Protoplasten festhalten an der Platte.</li>
	<li>Verwerfen Sie nach ein paar Sekunden die Kälberserum mit einer Pipette. Die resuspendierte Protoplasten in einen Brunnen der Kultur-Platte zu übertragen. Die Platte mit einem Deckel abdecken.</li>
</ol>5. Protoplasten Inkubation und Ernte
<ol><li>1-2 Tage im Dunkeln inkubieren Sie die Protoplasten bei Raumtemperatur. Hinweis: Wir fanden, dass zwei Tagen Inkubation stärkeren fluoreszierendes Protein-Signal in der Regel im Vergleich zu einem eintägigen Inkubation ergibt und das fluoreszierende Protein-Signal für bis zu 3-4 Tage dauern kann.</li>
	<li>Übertragen Sie die Protoplasten-Lösung auf einem 1,5 mL geringe Haftung Microcentrifuge Schlauch. Zentrifugieren Sie das Rohr 100 X g für 1-2 min bei Raumtemperatur Protoplasten zu ernten. Entfernen Sie den Überstand mit einer Pipette und übertragen Sie 10 µL Protoplasten auf einen Objektträger.</li>
	<li>Fluoreszenzsignal unter Fluoreszenz oder der konfokalen Mikroskopie zu beobachten. Verwenden Sie nicht-transformierten Protoplasten als Negativkontrolle (kein Signal sollte eingehalten werden).</li>
</ol>

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Die Autoren erklären, dass sie keine finanziellen Interessenkonflikte.

Dieses Protokoll für die Isolierung von Soja Protoplasten und die Anwendung auf transiente Expression Studien wurde gründlich getestet und funktioniert sehr gut in unserem Labor. Die Verfahren sind einfach und leicht und erfordern ordentliche Ausrüstung und minimalen Kosten. Unser Protokoll liefert große Mengen an gleichbleibende hohe Qualität Protoplasten im Vergleich zu bereits gemeldeten Methoden34,35,36,<sup ...

Protoplasten sind Pflanzenzellen, die Zellwände entfernt haben. Sie pflegen die meisten Funktionen und Aktivitäten von Pflanzenzellen, Protoplasten sind ein gutes Modellsystem zur Beobachtung und Auswertung der vielfältigen zelluläre Ereignisse, und wertvolle Tools für die somatische Hybridisierung1 zu studieren und Werk Regeneration2. Protoplasten wurden für Werk Verwandlung3,4,5, ebenfalls weit verbreitet genutzt, da Zellwände sonst den Durchgang von DNA in die Zelle blockieren würde. Protoplasten besitzen einige der physiologischen Reaktionen und zelluläre Prozesse der intakten Pflanzen, daher bietet Grundwert in der Grundlagenforschung, Protein subzelluläre Lokalisation6,7,8zu studieren, Protein-Protein Interaktionen9,10und Promotor-Aktivität11,12,13 in Leben Zellen.
Die Isolation der Pflanze Protoplasten wurde erstmals 196014 gemeldet und die Protokolle für Isolation und Transformation von Protoplasten entwickelt und optimiert wurden. Ein Standardverfahren der Protoplasten Isolation beinhaltet das Schneiden der Blätter und enzymatische Verdauung der Zellwände, gefolgt von Trennung von freigegebenen Protoplasten von Gewebe nicht verdaut Schutt. Transformationsstrategien umfasst Elektroporation15,16, Mikroinjektion17,18und Polyethylenglykol (PEG)4,5,19 Methoden. Eine Vielzahl von Arten gemeldet wurden erfolgreich für die Protoplasten Isolierung, einschließlich Zitrusfrüchte20, Brassica21, Solanaceae22 und anderen Zierpflanzen Familien23,24. Während verschiedene Gewebetypen in verschiedenen Arten verwendet werden, wurde ein System von transienten Expression in Arabidopsis Mesophyll Protoplasten (TEAMP) aus den Blättern der Modellpflanze Arabidopsis Thaliana isoliert gut etablierte25 und weit verbreiteten für vielfältige Anwendungen.
Sojabohne (Glycine max (L.) GESEG.) gehört zu den wichtigsten Protein und Öl Nutzpflanzen26. Im Gegensatz zu Arabidopsis und Reis transgene Sojapflanzen zu erhalten gilt eher schwierig und geringe Effizienz. Agrobacterium Tumefaciens-vermittelten Infiltration ist im Volksmund für transiente Genexpressionsstudien in der Epidermiszellen in Tabak27 und Sämlinge in Arabidopsis28,29, verwendet worden, während Transformation von behaarten Wurzeln in Soja30 Agrobacterium Rhizogenes bestritten. Virusbedingte Gen-Silencing-Ansätze wurden für Herabregulation von Ziel Gene31,32 und transiente Expression33 in einer systematischen Art und Weise genutzt. Protoplasten bieten eine wertvolle und vielseitige Alternative zu diesen Ansätzen. Protoplasten können Soja die oberirdische Materialien entnommen werden und ermöglichen schnelle und synchronisierte Transgene Ausdruck. Jedoch, seit die erste erfolgreiche Isolierung der Sojabohne Protoplasten in 198334gab es nur Berichte über die Anwendung von Protoplasten in Soja35,36,37, 38, in erster Linie aufgrund der relativ niedrigen Erträgen von Soja Protoplasten.
Hier beschreiben wir eine einfache und effiziente Protokoll für die Isolierung von Soja Protoplasten und seinen Antrag auf vorübergehende Genexpressionsstudien. Junge unifoliate Blätter aus Soja-Keimlingen konnten wir große Mengen an lebenswichtigen Protoplasten innerhalb weniger Stunden zu erhalten. Darüber hinaus haben wir einen PEG-Kalzium-vermittelten Transformationsmethode optimiert, die einfache und kostengünstige, DNA in Soja Protoplasten mit hohem Wirkungsgrad zu liefern ist.

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			<td>USA Scientific</td>
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			<td height="21" style="height:21px;width:256px;">EZNA plasmid miniprep kit</td>
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			<td height="21" style="height:21px;width:256px;">GeneJET Plasmid Miniprep Kit</td>
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			<td height="21" style="height:21px;width:256px;">Jencons Powerpette Plus Pipet Controller</td>
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			<td height="24" style="height:25px;width:256px;">Zeiss 710 Confocal Microscope</td>
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			<td height="21" style="height:21px;width:256px;">15 mL Centrifuge Tubes</td>
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			<td style="width:119px;">C1018-P</td>
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			<td height="21" style="height:21px;width:256px;">50 mL Centrifuge Tubes</td>
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			<td height="21" style="height:21px;width:256px;">Newborn Calf Serum</td>
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			<td height="21" style="height:21px;width:256px;">Soil</td>
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			<td height="21" style="height:21px;width:256px;">LB Broth, Lennox (Powder)</td>
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			<td style="width:119px;">BP1427-500</td>
			<td></td>
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a simple method for isolation of soybean protoplasts and application to transient gene expression analyses

Sojabohne (Glycine max (L.) GESEG.) ist eine wichtige Kulturarten und eine Hülsenfrucht-Modell für das Studium der genetischen und biochemischen Wege geworden. Daher ist es wichtig, eine effiziente transiente gen Expressionssystems in Soja zu etablieren. Hier berichten wir ein einfaches Protokoll für die Vorbereitung der Sojabohne Protoplasten und seinen Antrag auf vorübergehende Funktionsanalysen. Wir fanden, dass große Mengen an hochwertigen Protoplasten jungen unifoliate Blätter aus Soja-Keimlingen führte. Durch die Optimierung der PEG-Kalzium-vermittelten Transformationsmethode, erreichten wir hohe Transformationseffizienz mit Soja unifoliate Protoplasten. Dieses System bietet ein effiziente und vielseitige Modell für die Prüfung von komplexen regulatorischen und Signalisierung Mechanismen im live Soja Zellen und können helfen besser zu verstehen, diverse zelluläre, Entwicklungsstörungen und physiologische Prozessen von Leguminosen.

Verschiedene Organe des 10 - Tage alten Sojabohnen für Vorbereitung der Protoplasten (Abbildung 1) getestet wurden und Erträge beobachtet, unter dem Mikroskop (Abbildung 2). Zellwände aus Keimachse und Epicotyl waren kaum verdaut, und einige Zellen geblieben (Abbildung 2 b, 2 C) miteinander verbunden. Im Keimblatt (Abb. 2D) und Wurzel (<strong class="...

Watch this Scientific Journal Video about A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses at JoVE.com

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

Wir entwickelten ein einfaches und effizientes Protokoll für die Herstellung großer Mengen von Soja Protoplasten, komplexe regulatorische und Signalisierung Mechanismen in lebenden Zellen zu studieren.

Eine einfache Methode zur Isolierung der Sojabohne Protoplasten und Anwendung auf vorübergehende Genexpressionsanalysen

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. ...

a-simple-method-for-isolation-soybean-protoplasts-application-to

Research

JoVE Journal

Biology

24.4K Aufrufe.  California State University, Northridge. Wir entwickelten ein einfaches und effizientes Protokoll für die Herstellung großer Mengen von Soja Protoplasten, komplexe regulatorische und Signalisierung Mechanismen in lebenden Zellen zu studieren.

Serie: A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

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

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

RNA Isolation aus embryonalen Zebrafisch und cDNA-Synthese für die Genexpressionsanalyse

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

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

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

Hallo-C: eine Methode, um die dreidimensionale Architektur des Genoms Study.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

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 ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Isolierung von Ribosom gebunden Nascent Polypeptide<em&gt; In-vitro-</em&gt; Identifizieren Translational Pause Standorten entlang mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

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 ...

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR).

In vivo transfection of naked DNA by hydrodynamic gene delivery introduces genes into the tissue of an animal with minimal inflammatory response. Sufficient amounts of gene product are generated such that gene function and regulation as well as protein structure and function can be analyzed.

Transiente Expression von Proteinen durch Hydrodynamische Gentransfer in Mäusen

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...