Whitehead Institute Fellows Funds

Teil 1: cDNA-Synthese aus RNA <ol><li> Vor der Verwendung der Ambion Amino Allyl MessageAmp II Kit, fügen Sie die empfohlene Menge von 100% Ethanol zu dem Waschpuffer. </li><li> In PCR-Gefäß, fügen Sie zwischen 100 ng bis 5μg Gesamt-RNA und 1μl der T7-Oligo (dT)-Primer. Bringen Sie die Lautstärke von bis zu 12μl mit Nuklease-freiem Wasser. </li><li> Inkubieren Proben bei 70 ° C für 10 min in einem Thermocycler. </li><li> Entfernen RNA-Proben von 70 ° C und kurz zentrifugieren. Auf Eis legen. </li><li> Bereiten Sie die 1. Strand Synthesis Master-Mix und halten bei Raumtemperatur (mit 10% Überschuss zum Pipettieren Fehler). (Tabelle 1) </li><li> Gently Pipette Master Mix-oder Springmesser, zu mischen, und dann kurz zentrifugieren. </li><li> Transfer 8μl des Master Mix zu jeder RNA-Probe. Mix von Auf-und Abpipettieren 3-4 mal. </li><li> Inkubation bei 42 ° C für 2 Stunden in Thermocycler. </li><li> Centrifuge Proben kurz auf Eis stellen. Unmittelbar mit dem nächsten Schritt der dsDNA Synthese fort. </li><li> Bereiten Sie die 2 nd Strand Synthesis Master-Mix auf Eis (mit 10% Überschuss zum Pipettieren Fehler). (Tabelle 2) </li><li> Gently Pipette Master Mix-oder Springmesser, zu mischen, und dann kurz zentrifugieren. </li><li> Transfer-80μl jeder Probe und sanft durch Auf-und Abpipettieren 3-4 mal zu mischen. </li><li> Inkubation bei 16 ° C für 2 Stunden in Thermocycler. </li><li> Nach dem 2 Stunden Inkubation mit der cDNA clean-up Schritt fort oder einfrieren sofort bei -20 ° C. </li></ol> Teil 2: Double-cDNA clean-up <ol><li> Entfernen Sie die cDNA pur aus dem Kühlschrank und lassen Sie ihn auf Raumtemperatur für 30 Minuten vor dem Einsatz ausgleichen. Schütteln Sie die Flasche voll resuspendieren der magnetischen cDNA verbindliche Perlen vor der Verwendung. </li><li> Aliquot Nuklease-freiem Wasser in ein 1,5 ml Röhrchen und inkubieren bei 50-60 ° C für mindestens 10 Minuten während der letzten 2 Std. Inkubation. </li><li> Fügen Sie 180μl der cDNA pur zu jeder Probe und gründlich durch Auf-und Abpipettieren mischen. Übertragen Sie die Proben auf eine 96-Well-Rundboden-Platte. </li><li> Weiter zu den Proben durch leichtes Schütteln der Platte auf einem Schüttler für mindestens 2 Minuten mischen. </li><li> Bewegen Sie die Platte zu einem magnetischen stehen, um die magnetischen Kügelchen zu erfassen. Lassen Platte auf den Ständer für ca. 6 Minuten, oder bis die Mischung wird transparent und die Bindung Perlen haben pelletiert. </li><li> Vorsichtig absaugen der Überstand mit einem Vakuum-Sauger, ohne die magnetischen Kügelchen. Alternativ vorsichtig den Überstand mit einer Pipette und den Überstand verwerfen. </li><li> Die Platte aus dem magnetischen stehen. </li><li> Add 150 &amp;mgr; l cDNA Waschpuffer in jede Kavität der Platte für 1 Minute auf dem Schüttler bei mäßiger Geschwindigkeit. Perlen werden zu diesem Schritt zu zerstreuen, wegen der niedrigen Oberflächenspannung des Waschpuffer. </li><li> Bewegen Sie die Platte zu einem magnetischen stehen, um die magnetischen Kügelchen zu erfassen. </li><li> Vorsichtig absaugen der Überstand mit einem Vakuum-Sauger, ohne die magnetischen Kügelchen. Alternativ vorsichtig den Überstand mit einer Pipette und den Überstand verwerfen. </li><li> Die Platte aus dem magnetischen stehen. </li><li> Wiederholen Sie die Wäsche ein 2. Mal mit 150 &amp;mgr; l cDNA-Waschpuffer. </li><li> Nach dem 2. waschen, trocknen die Kügelchen durch Schütteln der Platte für 2 Minuten auf dem Schüttler bei maximaler Geschwindigkeit. Nicht übertrocknen! </li><li> Eluieren der cDNA von den Perlen, indem 18μl der vorgewärmten Nuklease-freies Wasser zu jeder Probe. </li><li> Kräftig schütteln, die Platte für 3 Minuten auf dem Schüttler, dann überprüfen Sie, ob die magnetischen Beads vollständig verteilt sind. Wenn nicht, weiter zittern. </li><li> Nachdem die magnetischen Beads vollständig zerstreut haben, bewegen Sie die Platte zu einem magnetischen stehen, um die magnetischen Kügelchen zu erfassen. </li><li> Sorgfältig Übertragung der eluierten cDNA (~ 16μl) in ein neues PCR-Platte (oder PCR-Röhrchen). </li><li> Gehen Sie direkt zum nächsten Schritt, oder frieren die cDNA bei -20 ° C. </li></ol> Teil 3: In-vitro-Transkription (IVT) <ol><li> Bereiten Sie das IVT Master-Mix bei Raumtemperatur. (Tabelle 3) </li><li> Gently Pipette die Master Mix-oder Springmesser, zu mischen und kurz zentrifugieren. </li><li> Add 24μl jeder Probe und sanft durch Auf-und Abpipettieren 3-4 mal zu mischen. </li><li> Bei 37 ° C für 14 Stunden in einem Thermocycler, dann bei 4 ° C bis bereit für den nächsten Schritt zu halten. </li></ol> Teil 4: aRNA clean-up nach IVT <ol><li> Vortex die RNA-Bindung Perlen kurz, um eine gleichmäßige Mischung vor Gebrauch zu erhalten. </li><li> Bereiten Sie die aRNA Binding Mix bei Raumtemperatur (Tabelle 4) Das vor der Zeit getan werden kann -. Die vorbereitete verbindliche Mischung kann bei Raumtemperatur gelagert werden bis zu einer Woche. </li><li> Gut mischen durch Vortexen. </li><li> Aliquot der aRNA Elutionspuffer in ein 1,5 ml Röhrchen und inkubieren bei 50-60 ° C für mindestens 10 Minuten. </li><li> Fügen Sie 70μl der aRNA verbindliche Mixzu jeder Probe und mischen Sie gut durch Auf-und Abpipettieren 3-4 mal. </li><li> Übertragen Sie die Proben aus der PCR-Platte, eine 96-Well-Rundboden-Platte. </li><li> Add 50 ul 100% Isopropanol zu jeder Probe und mischen Sie gut durch Auf-und Abpipettieren 3-4 mal. </li><li> Schütteln Sie die Platte auf einem Schüttler für mindestens 2 Minuten gründlich mischen der Proben. </li><li> Bewegen Sie die Platte zu einem magnetischen stehen, um die magnetischen Kügelchen zu erfassen. Lassen Sie die Platte auf dem Stand, bis die Mischung wird transparent und die Bindung Perlen haben pelletiert. </li><li> Vorsichtig absaugen der Überstand mit einem Vakuum-Sauger, ohne die magnetischen Kügelchen. Alternativ vorsichtig den Überstand mit einer Pipette und den Überstand verwerfen. </li><li> Die Platte aus dem magnetischen stehen. </li><li> Add 100 &amp;mgr; aRNA Waschlösung in jede Kavität der Platte für 1 Minute auf dem Schüttler bei mäßiger Geschwindigkeit. Perlen können nicht vollständig in diesem Schritt zu zerstreuen. </li><li> Bewegen Sie die Platte zu einem magnetischen stehen, um die magnetischen Kügelchen zu erfassen. </li><li> Vorsichtig absaugen der Überstand mit einem Vakuum-Sauger, ohne die magnetischen Kügelchen. Alternativ vorsichtig den Überstand mit einer Pipette und den Überstand verwerfen. </li><li> Die Platte aus dem magnetischen stehen. </li><li> Wiederholen Sie die Wäsche ein 2. Mal mit 100 &amp;mgr; aRNA Waschlösung. </li><li> Nach dem 2. waschen, trocknen die Kügelchen durch Schütteln der Platte für 1 Minute auf dem Schüttler bei maximaler Geschwindigkeit. Nicht übertrocknen Proben! </li><li> Eluieren aRNA von den Perlen, indem 40 ul vorgewärmte aRNA Elutionspuffer zu jeder Probe. </li><li> Kräftig schütteln, die Platte auf dem Schüttler für 3 Minuten, dann überprüfen Sie, ob die magnetischen Beads vollständig verteilt sind. Wenn nicht, weiter zittern. </li><li> Nachdem die magnetischen Beads vollständig zerstreut haben, bewegen Sie die Platte zu einem magnetischen stehen, um die magnetischen Kügelchen zu erfassen. Der Überstand enthält das gereinigte-up Amino-allyl eingebaut aRNA. </li><li> Sorgfältig Übertragung der eluierten aRNA einen neuen PCR-Platte (oder PCR-Röhrchen). </li><li> (Optionaler Schritt) Überprüfen Sie die RNA-Konzentration der Proben durch die Messung 1.5μl auf einem NanoDrop Spektralphotometer. </li><li> Unmittelbar auf den nächsten Schritt fort, oder lagern Sie das aRNA bei -80 ° C. </li></ol> Tabelle 1: cDNA 1 M. Strand Synthesis Master Mix <table border="1" cellpadding="8"><tbody><tr><td> Reagens </td><td> Betrag für 1-Reaktion </td></tr><tr><td> 10X First Strand Buffer </td><td> 2 ul </td></tr><tr><td> dNTP Mix </td><td> 4 ul </td></tr><tr><td> Ribonuclease Inhibitor </td><td> 1 ul </td></tr><tr><td> Array Script </td><td> 1 ul </td></tr></tbody></table> Tabelle 2: cDNA 2. Strand Synthesis Master Mix <table border="1" cellpadding="8"><tbody><tr><td> Reagens </td><td> Betrag für 1-Reaktion </td></tr><tr><td> Nuclease freies Wasser </td><td> 63 ul </td></tr><tr><td> 10X Zweiten Strand Buffer </td><td> 10 pl </td></tr><tr><td> dNTP Mix </td><td> 4 ul </td></tr><tr><td> DNA Polymerase </td><td> 2 ul </td></tr><tr><td> RNase H </td><td> 1 ul </td></tr></tbody></table> Tabelle 3: IVT Master Mix <table border="1" cellpadding="8"><tbody><tr><td> Reagens </td><td> Betrag für 1-Reaktion </td></tr><tr><td> aaUTP-Lösung (75 mM) </td><td> 2 ul </td></tr><tr><td> ATP, CTP, GTP-Mix (25 mM) </td><td> 12 ul </td></tr><tr><td> T7 UTP-Lösung (75 mM) </td><td> 2 ul </td></tr><tr><td> T7 10X Reaktionspuffer </td><td> 4 ul </td></tr><tr><td> T7-Enzym-Mix </td><td> 4 ul </td></tr></tbody></table> Tabelle 4: aRNA Binding Mix <table border="1" cellpadding="8"><tbody><tr><td> Reagens </td><td> Betrag für 1-Reaktion </td></tr><tr><td> RNA Binding Perlen * </td><td> 10 pl </td></tr><tr><td> Bead Resuspension Solution * </td><td> 4 ul </td></tr><tr><td> 100% Isopropanol ** </td><td> 6 ul </td></tr><tr><td> aRNA Binding Buffer Concentrate </td><td> 50 pl </td></tr></tbody></table> * Mix der RNA-Bindung Perlen mit der Raupe Resuspension Lösung zuerst. ** Fügen Sie das Isopropanol und gut mischen, bevor Sie die aRNA Bindungspuffer konzentrieren.

<ol>
	<li>Rubins, K. H., Hensley, L. E., Bell, G. W., Wang, C., Lefkowitz, E. J., Brown, P. O., Relman, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+viral+gene+expression+programs+during+poxvirus+infection:+a+transcriptional+map+of+the+vaccinia+and+monkeypox+genomes.">Comparative analysis of viral gene expression programs during poxvirus infection: a transcriptional map of the vaccinia and monkeypox genomes.</a> PLoS ONE. 3 (7), e2628-e2628 (2008).</li><li>Assarsson, E., Greenbaum, J. A., Sundstr&#246;m, M., Schaffer, L., Hammond, J. A., Pasquetto, V., Oseroff, C., Hendrickson, R. C., Lefkowitz, E. J., Tscharke, D. C., Sidney, J., Grey, H. M., Head, S. R., Peters, B., Sette, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+analysis+of+a+complete+poxvirus+transcriptome+reveals+an+immediate-early+class+of+genes.">Kinetic analysis of a complete poxvirus transcriptome reveals an immediate-early class of genes.</a> Proc. Natl. Acad. Sci. 105 (6), 2140-2145 (2008).</li><li>Satheshkumar, P. S., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poxvirus+transcriptome+analysis.">Poxvirus transcriptome analysis.</a> Proc. Natl. Acad. Sci. U.S.A. 105, E62-E62 (2008).</li><li>Assarsson, E., Greenbaum, J. A., Sundstr&#246;m, M., Schaffer, L., Hammond, J. A., Pasquetto, V., Oseroff, C., Hendrickson, R. C., Lefkowitz, E. J., Tscharke, D. C., Sidney, J., Grey, H. M., Head, S. R., Peters, B., Sette, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A.+Reply+to+Satheshkumar+and+Moss:+Poxvirus+transcriptome+analysis.">A. Reply to Satheshkumar and Moss: Poxvirus transcriptome analysis.</a> Proc. Natl. Acad. Sci. U.S.A. 105, E63-E64 (2008).</li><li>Guerra, S., L&#243;pez-Fern&#225;ndez, L. A., Pascual-Montano, A., Mu&#241;oz, M., Harshman, K., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+gene+expression+survey+of+vaccinia+virus+infection+of+human+HeLa+cells.">Cellular gene expression survey of vaccinia virus infection of human HeLa cells.</a> J Virol. 77 (11), 6493-6506 (2003).</li><li>Guerra, S., L&#243;pez-Fern&#225;ndez, L. A., Conde, R., Pascual-Montano, A., Harshman, K., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+analysis+reveals+characteristic+changes+of+host+cell+gene+expression+in+response+to+attenuated+modified+vaccinia+virus+Ankara+infection+of+human+HeLa+cells.">Microarray analysis reveals characteristic changes of host cell gene expression in response to attenuated modified vaccinia virus Ankara infection of human HeLa cells.</a> J Virol. 78 (11), 5820-5824 (2004).</li><li>Guerra, S., L&#243;pez-Fern&#225;ndez, L. A., Pascual-Montano, A., N&#225;jera, J. L., Zaballos, A., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+response+to+the+attenuated+poxvirus+vector+NYVAC:+upregulation+of+apoptotic+genes+and+NF-kappaB-responsive+genes+in+infected+HeLa+cells.">Host response to the attenuated poxvirus vector NYVAC: upregulation of apoptotic genes and NF-kappaB-responsive genes in infected HeLa cells.</a> J Virol. 80 (2), 985-998 (2006).</li><li>Guerra, S., N&#225;jera, J. L., Gonz&#225;lez, J. M., L&#243;pez-Fern&#225;ndez, L. A., Climent, N., Gatell, J. M., Gallart, T., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+gene+expression+profiling+after+infection+of+immature+human+monocyte-derived+dendritic+cells+by+the+attenuated+poxvirus+vectors+MVA+and+NYVAC.">Distinct gene expression profiling after infection of immature human monocyte-derived dendritic cells by the attenuated poxvirus vectors MVA and NYVAC.</a> 81 (16), 8707-8721 (2007).</li></ol>

The authors have nothing to disclose.

Kritische Schritte Eine zweite Runde der Amplifikation wird nicht empfohlen, da Verzerrungen in Array-Daten beobachtet wurden. Mischen sorgfältig an jedem enzymatischen Schritt (1 st und 2 nd cDNA-Synthese, IVT) ist entscheidend für die Erzielung einer guten Verstärkung liefert, wie die Inkubation von jedem enzymatischen Schritt bei der entsprechenden Temperatur. Ein PCR-Cycler mit verstellbaren beheizten Deckel wird bevorzugt - auch Abweichungen so klein wie 2-3 Grad ...

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		<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>Amino Allyl MessageAmp II aRNA amplification kit</td>
<td>Reagent</td>
<td>Applied Biosystems</td>
<td>AM1753</td>
<td>For 20 reactions</td>
</tr>
<tr>
<td>Amino Allyl MessageAmp II aRNA amplification kit</td>
<td>Reagent</td>
<td>Applied Biosystems</td>
<td>AM1821</td>
<td>For 100 reactions, in a 96-well format</td>
</tr>
<tr>
<td>NanoDrop ND-1000 UV-VIS spectrophotometer</td>
<td>Other</td>
<td>NanoDrop</td>
<td>ND-1000</td>
<td>Or equivalent spectrophotometer</td>
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vaccinia virus infection &amp; temporal analysis of virus gene expression: part 2

Die Familie<em&gt; Poxviridae</em&gt; Besteht aus großen doppelsträngigen DNA-Viren enthalten, die ausschließlich im Zytoplasma der infizierten Zellen replizieren. Die Mitglieder des<em&gt; Orthopox</em&gt; Gattung gehören Variola, dem Erreger der menschlichen Pocken, Affenpocken und Vaccinia (VAC), der prototypische Mitglied der Virus-Familie. Innerhalb der relativ groß (~ 200 kb) Vaccinia-Genom gibt drei Klassen von Genen kodiert: frühe, mittlere und spät. Während alle drei Klassen von viral-kodierten RNA-Polymerasen transkribiert werden, dient jede Klasse eine andere Funktion im Lebenszyklus des Virus. Pockenviren nutzen mehrere Strategien zur Modulation der zellulären Umgebung Host während der Infektion. Um Regulierung der Wirt und Virus Genexpression verstehen, müssen wir genomweite Ansätze genutzt werden, um Transkript Fülle von beiden Viren und Wirtszellen zu analysieren. Hier zeigen wir, zeitlicher Verlauf Infektionen von HeLa-Zellen mit Vaccinia-Virus und Sampling-RNA zu verschiedenen Zeitpunkten nach der Infektion. Beide Gastgeber und virale RNA isoliert und verstärkt für die Hybridisierung an Microarrays zur Analyse der Genexpression.

Watch this Scientific Journal Video about Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 2 at JoVE.com

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 2

Protokoll zur Vaccinia-Infektion von HeLa-Zellen und die Analyse von Host-und virale Genexpression.

Vaccinia Virus Infection &amp; Temporal Analysis des Virus Gene Expression: Teil 2

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells.  Members of ...

vaccinia-virus-infection-temporal-analysis-virus-gene-expression-part

Research

JoVE Journal

Biology

8.0K Aufrufe.  MIT - Massachusetts Institute of Technology. Protokoll zur Vaccinia-Infektion von HeLa-Zellen und die Analyse von Host-und virale Genexpression.

Serie: Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 2

Bacterial Gene Expression Analysis Using Microarrays

Bakterielle Gene Expression Analysis Using Microarrays

Forschung

Biologie

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 ...

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 1

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 1 of 3.

Vaccinia Virus Infection &amp; Temporal Analysis des Virus Gene Expression: Teil 1

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 3

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 3 describes the process of fluorescently labeling the amplified RNA from both host and viral samples by amino allyl coupling of dyes. Part 3 of 3.

Vaccinia Virus Infection &amp; Temporal Analysis des Virus Gene Expression: Teil 3

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

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 ...

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 ...

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

Agrobacterium-vermittelte Virus-induced gene silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

Die MultiBac Protein Complex Produktion Platform am EMBL

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

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 ...

Methods Article

Vaccinia Virus Infection & Temporal Analysis des Virus Gene Expression: Teil 2