Thanks to Brad Lackford for assistance optimizing some of the techniques described in this manuscript.

1. Aufrechterhaltung der Makrophagenzellinien <ol><li> Wachsen RAW264.7 und J774A.1 Zelllinien in DMEM, ergänzt mit 10% fötalem Rinderserum (FBS) bei 37 ºC in Gegenwart von 5% CO 2. Zur Aufrechterhaltung der Sterilität wird mit 1% Penicillin / Streptomycin (Pen / Strep) an die Medien (obwohl dies nicht unbedingt erforderlich). </li><li> Critical Schritt: Stellen Sie sicher, dass die Makrophagen werden in einem gesunden Zustand wächst für eine effiziente Transfektion und siRNA-induzierte Gen Knockdown. Verwenden Zellen zwischen den Passagen 3 und 10 nach frischen Auftauen, da diese Makrophagen Linien zeigen die besten Transfektionseffizienz und pflegen robust nach der Transfektion Überleben in diesem Stadium; nicht mit einem höheren Durchlaufhäufigkeit (&gt; 20), als Transfektionseffizienz und Knock-down sowohl stark vermindert. </li><li> Für eine optimierte siRNA Transfektion Platte Zellen bei 20% Konfluenz und wachsen ein bis zwei Tage, bis die Zellen nicht mehr als 80% Konfluenz erreichen. Overgrown Zellen nicht transfizieren efficiently. </li></ol> 2. Programmierung der 96-Loch-Shuttle System zur Transfektion <ol><li> Startet den 96-well-Shuttle-Software auf dem Computer mit dem Shuttle verbunden. Wählen Sie neue Parameterdatei von der oberen linken Dateimenü. </li><li> Markieren der Vertiefungen, die auf der angezeigten Platte mit 96 Vertiefungen schema transfiziert werden. Ein grünes Feld zeigt an, welche Brunnen ausgewählt. </li><li> Wählen Sie das Programm, das auf die Makrophagen-Zelllinie verwendet (DS-136 für RAW264.7 oder DS-130 für J774A.1), und wählen gelten, unter dem diagramed 96-Well-Platte befindet entspricht. Beachten Sie, dass nach der Programmwahl, die Vertiefungen auf der schematischen Platte wird dunkelblau eingefärbt werden. Leere Kreise zeigen Brunnen, die nicht ein Programm zugewiesen haben und nicht schockiert sein. </li></ol> 3. Vorbereitung der Reagenzien für die Transfektion <ol><li> Herstellung der Arbeits Nucleofector ™ Lösung durch Mischen SF-Zelllinie mit 96 Vertiefungen Nucleofector ™ Lösungmit dem Inhalt des beiliegenden Ergänzungsrohr. Ermöglichen, dass die gemischten Reagenzien bei Raumtemperatur vor der Verwendung äquilibriert. Speichern überschüssige komplette Nucleofector ™ Lösung bei 4 ° C für bis zu 3 Monate für die zukünftige Verwendung. </li><li> Vorwärmen der DMEM, RPMI-1640, 0,25% Trypsin / EDTA und Phosphat-gepufferter Salzlösung (PBS) bei 37 ºC vor der Herstellung der zur Transfektion Makrophagen. </li><li> Bereiten Sie die siRNA zur Transfektion durch Auftauen auf Eis. Um die Anzahl der Frost taut der siRNA minimieren, Gefriert neue siRNAs in Aliquots sie das erste Mal verwendet werden. </li><li> In ersten Experimenten verwendet siRNA in einer relativ hohen Dosis von 2 &amp; mgr; M (1:10 verdünnt 20 &amp; mgr; M Bestände). Anschließend titriert siRNAs, die robust Gen auf niedrigere Dosen Knockdown zu induzieren, obwohl typischerweise höhere Dosen sind für eine effiziente Gen Knockdown in Makrophagen im Vergleich zu anderen Zelltypen benötigt. HINWEIS: siRNAs sind aus vielen Quellen erhältlich; recht robust Knock-down kann erhalten werdenVerwendung siGENOME SMARTPOOLs, die Pools von vier siRNAs jedes Gen sind. Als negative Kontrolle, verwenden Sie eine verschiedene nicht-Targeting siRNA Pools oder siRNAs. </li><li> Zur Überwachung der Transfektionseffizienz, Transfektion entweder die pmaxGFP Kontrollplasmid (in den Amaxa Kits bereitgestellt) oder fluoreszenzmarkierten siRNAs parallel zu den experimentellen siRNAs. 30-50% erwartet Transfektion des GFP-Plasmid (durch Mikroskopie getestet oder Durchflusszytometrie Tag nach der Transfektion) und&gt; 99% der Transfektion von fluoreszenzmarkierten siRNA (durchflusszytometrisch unmittelbar nach der Transfektion und Gewinnung getestet). </li></ol> 4. Vorbereiten der Makrophagen für die Transfektion <ol><li> Trennen adhärenten Makrophagen aus den Zellkulturschalen durch Trypsinierung. Saugen Sie die Medien und waschen Sie die Zellen zweimal mit 10 ml PBS. Nach Entfernen des PBS, inkubiert die Zellen mit vorgewärmtem 0,25% Trypsin / EDTA (1 ml / 10 cm Platte) für 1-2 min, bis die Zellen beginnen, abzunehmen. Die Platte vorsichtig antippen und verschieben Sie die Lösungention während der Inkubation zu Trypsinierung maximieren. </li><li> Nach der Inkubation hinzufügen 9 ml DMEM + FBS Medien, vorsichtig mischen auf und ab, und sammeln die Zellen durch Pipettieren in einem sterilen Röhrchen. </li><li> Zähle die Anzahl der isolierten Makrophagen mit einer Zählkammer. Erwarten Sie etwa 10 Millionen Makrophagen pro 10 cm Platte. Berechnung der Gesamtzahl von Makrophagen erforderlich ist; jede Vertiefung der 96-Well-Shuttle erfordert 200.000 Zellen. Denken Sie daran, im Wert von Zellen, die ein paar zusätzliche Brunnen &#39;in den zum Pipettieren Verluste ermöglichen. </li><li> Pipettieren die berechnete Anzahl von Zellen in ein Zentrifugenröhrchen. Zentrifugieren der Zellen für 10 min bei 150 xg bei Raumtemperatur. Zu schnell Zentrifugieren Sie keine, da dies Makrophagen Gesundheit und Transfektionseffizienz verringern. </li></ol> 5. Nucleofection von Makrophagen mit siRNA <ol><li> Mit einer sterilen 96-Well-Rundbodenplatte (das Mischen erleichtert und minimiert Reagenzienverlust), fügen Sie 2 ul jeder siRNA in jede Vertiefung zu sein transfreflektierte. Hinweis: Je nach der Anzahl der siRNAs, kann dies während des Makrophagen Zentrifugationsschritt durchgeführt werden. </li><li> Saugen Sie das Medium aus dem zentrifugiert Zellen. Erythrozyten vorsichtig resuspendieren in Nucleofector ™ -Lösung SF (mit dem Ergänzungslösung), unter Verwendung von 20 &amp; mgr; l pro 200.000 Zellen pelletiert. Sobald resuspendiert, verwenden Sie die Zellen sofort für eine optimale Transfektion. </li><li> Übertragen resuspendierten Zellen in ein steriles Trog; mit einer Mehrkanalpipette, Transfer 20 ul der resuspendierten Zellgemisch in jede Vertiefung der 96-Well-Platte mit den siRNAs. Vorsichtig mischen oben und unten. </li><li> Transfer 20 ul der Zell / siRNA-Gemisch in die 96-Well-Platte Nucleocuvette ™ kritischer Schritt. Erzeugen von Blasen zu vermeiden, während der Übertragungsschritt so kann Fehler während Nucleofection induzieren. </li><li> Nach dem Platzieren aller Versuchsproben in der Transfektion Platte Bedeckung die 96-well Platte mit dem Deckel versehen und very klopfen Sie leicht auf eine harte Oberfläche, um alle Blasen aus den Proben zu entfernen. </li><li> Zeigen 96-Well-Platte mit Deckel in den 96-Loch-Nucleofector Shuttle und wählen Sie &quot;laden und starten&quot; auf der rechten unteren Ecke des Programmbildschirm. Vor Nucleofection, folgen Sie dem Programm Aufforderung, um die Ergebnisse zu speichern. Hinweis: Während der Nucleofection Prozess, Brunnen erfolgreich transfiziert werden auf der 96-Loch-auf dem Bildschirm schema durch einen grünen Kreis mit einem Pluszeichen dargestellt werden. Ein roter Kreis mit Minuszeichen zeigt einen Fehler an, höchstwahrscheinlich aufgrund von Blasen oder Fehl Pipettieren das richtige Volumen von Reagenzien. </li></ol> 6. Wiederherstellung und Oberflächen von Makrophagen <ol><li> Unmittelbar nach Nucleofection, mit einer Mehrkanalpipette je 80 ul vorgewärmten (37 ° C) RMP1-1640 Medien in jede Vertiefung. Fügen Sie die Medien auf der Seite jeder gut, so dass die Medien langsam mit den transfizierten Zellen, die sehr empfindlich sind in dieser Stufe mischen. Haben die Medien nicht zu schnell, da diesstark vermindern Lebensfähigkeit. </li><li> Platzieren Sie die Platten mit 96 Vertiefungen transfiziert mit Proben und RPMI-1640 in einem 37 ºC-Inkubator mit 5% CO 2 für 2 min; Dies ist wichtig, damit die Zellen sich zu erholen, bevor Manipulation weiter. </li><li> Entfernen Sie den 96-Well-Platte aus dem Inkubator und die Mischung vorsichtig übertragen aus jeder Vertiefung in eine 96-Well-Gewebekulturplatte mit einer Mehrkanalpipette. </li><li> Mit einer Mehrkanalpipette, 100 l vorgewärmten (37 ° C) DMEM + FBS-Medium in jede Vertiefung. </li><li> Inkubieren des 96-Well-Gewebekulturplatte mit Proben in einem 37 ºC-Inkubator mit 5% CO 2 für 24-36 h (Optimierung der genaue Zeitpunkt für die einzelnen siRNAs, wobei diese Zeitbereich arbeitet im allgemeinen gut). </li></ol> 7. Die Stimulation der Makrophagen mit LPS <ol><li> Im Anschluss an die 24 bis 36 Stunden nach der Nucleofection Inkubation vorbereiten LPS (oder andere PAMPs) in frischem Medium. Bereiten Sie genügend frisches Medium für alle Vertiefungen (200 ul medien / Vertiefung der 96-Well-Platte plus ein kleines Extra für Pipettieren). Verdünnte LPS in einer Endkonzentration von 20 ng / ml in DMEM + FBS. </li><li> Saugen Sie vorsichtig Medien aus dem 96-Well-Platte und fügen Sie die frische Medien mit den LPS zu den Zellen. </li><li> Überwachung der Reaktion auf LPS oder andere PAMPs zu verschiedenen Zeitpunkten nach der Stimulation. 6 Stunden ausreichend, um eine robuste inflammatorische Zytokin-Antwort für Cytokine wie IL-6 oder TNF &amp; alpha; zu erzeugen. </li></ol> 8. Überwachung der induzierten angeborenen Immunantwort, Knock-down und Lebensfähigkeit in Makrophagen <ol><li> LPS-induzierte Cytokin-Produktion zu überwachen, Pipetten den Überstand von den Zellen in eine 96-Well Platte. Cytokin-Produktion kann dann durch ELISA gemessen werden. </li><li> Nachdem der Überstand entfernt wird, überwacht die Zelllebensfähigkeit mit Fluorescein-Diacetat (wenn durch zelluläre Esterasen in lebenden Zellen gespalten wird, wird diese Verbindung fluoreszierend). Identifizieren siRNAs, die Zielgene, die die Lebensfähigkeit der Zellen zu steuern mit tseinen Ansatz. Hinweis: Die Transfektion Prozess selbst sollte eine geringe Wirkung auf die Lebensfähigkeit haben, wenn sie richtig durchgeführt wird. <ol><li> In Fluoresceindiacetat (5 mg / ml Stamm in Aceton) in jede Vertiefung (1: 100 endg Verdünnung in PBS). </li><li> Inkubieren bei Raumtemperatur für ein bis fünf Minuten, und anschließend die Fluoreszenz in den Zellen unter Verwendung eines Plattenlesegerät (460 nm Anregung, 515 nm Emission) zu überwachen. </li><li> Optional: Um sicherzustellen, dass die Fluoreszenz in den Vertiefungen ist einheitlich, lysieren die Zellen mit Puffer wie RIPA; Dies stellt eine gleichförmige Fluoreszenzsignal in dem Bohrloch, wie einige Plattenleser so konfiguriert sind, nur einen kleinen Teil des Schachtes zu überwachen; Wenn die Zellen nicht gleichförmig überzogen, könnte das Fluorescein-Diacetat ein ungenaues Lesen zu geben. </li></ol></li><li> Als eine Alternative zur Überwachung Zelllebensfähigkeit, überwachen siRNA-induzierte Gen-Knockdown von qPCR unter Verwendung von RNA aus den anhaftenden Zellen. <ol><li> Nachdem der Überstand wird von den Zellen entfernt (Schritt 8.1), Add RLT-Puffer (die Lyse und Konservierung von RNA verwendet wird) direkt auf die anhaftenden Zellen, um sie zu lysieren. </li><li> Isolieren RNA mit einem RNA Vorbereitung Kit wie die RNeasy Mini-Kit, und verwenden qPCR mit Gen-spezifischen Primern, um Gen-Knockdown relativ zu siRNA Behandlung steuern überwachen. Verwenden Primer an Βactin oder GAPDH zur Normalisierung. </li></ol></li><li> Als eine weitere Alternative zu anderen Aspekten der angeborenen Immunantwort zu überwachen, überwacht die phagozytische Fähigkeit der Makrophagen durch Zugabe von FITC-markierte E. coli Partikel direkt auf die anhaftenden Zellen unter Verwendung eines Kits. Nach Herstellerprotokoll, das nur ein paar Stunden dauert, zu überwachen Phagozytose mit einem Plattenleser oder Mikroskopie. </li></ol>

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Die Autoren erklären, dass sie keine konkurrierenden finanziellen Interessen. Dieser Artikel wurde von Lonza, Inc. gesponsert

Es wurden zahlreiche Studien veröffentlicht, in denen einzelne Gene von siRNA in murinen Makrophagen gezielt worden. Während Lipid-vermittelte Transfektion wurde verwendet, um siRNA zur Makrophagenzelllinien auf einer individuellen Basis zu liefern, leiden diese Verfahren unter potentiellen Auswirkungen auf die Lebensfähigkeit, begrenzte Knock-down, und die Variabilität von Gen zu Gen. Um robustere Assays, die verwendet werden könnten, um Gene in Mittel- oder Hochdurchsatzverfahren zielen entwickeln optimierten wir...

In diesem Protokoll beschreiben wir effiziente Verfahren, um Gene in Maus-Makrophagen-Zelllinien unter Verwendung von siRNAs zu hemmen und die Wirkung dieser Behandlungen auf die angeborene Immunantwort zu überwachen. Diese Verfahren werden in 96-Well-Format durchgeführt, so dass für RNAi-Screens in Mittel- oder Hochdurchsatz-Mode. In Reaktion auf eine Infektion, montieren Menschen sofort angeborenen Immunantwort und eine langsamere, aber bestimmte adaptive Immunantwort. Diese schnelle angeborenen Immunantwort beinhaltet die Rekrutierung und Aktivierung von phagozytierenden angeborenen Immunzellen einschließlich Makrophagen ein. Klassisch aktivierte Makrophagen sind im akuten Entzündungsreaktionen beteiligt und produzieren antimikrobielle Proteine ​​und Verbindungen, Zytokine und Chemokine, und präsentieren Antigene 2,3. Alternativ aktivierte Makrophagen spielen eine Rolle bei der Regulierung der Immunität, die Aufrechterhaltung der Toleranz, die Gewebereparatur und Wundheilung 4-8. Aufgrund ihrer breiten Palette von Funktionen, Makrophagen eine Rolle bei zahlreichen Krankheiten, darunter Arteriosklerose, Arthritis und Krebs 9 spielen. Somit hat das Studium der Makrophagen für einige Zeit in einer Vielzahl von Krankheitsfelder ein Schlüsselbereich der Forschung. Trotz ihrer großen Bedeutung in der angeborenen Immunantwort kann Makrophagen werden herausfordernde Zellen, mit zu arbeiten. Insbesondere ist es schwierig, eine effiziente Transfektion mit Lipidreagenzien in Makrophagen ohne assoziierte Toxizität 10,11 erhalten. Außerdem, selbst wenn siRNA wirksam an Makrophagen geliefert, die Robustheit der RNAi-induzierten Gens oft Knockdown kann ziemlich moderat sein und kann von Gen zu Gen variieren. Um diese technischen Herausforderungen zu überwinden, haben wir Transfektion und Zuschlagsverfahren 12-16 in zwei Maus-Makrophagen-Zelllinien, RAW264.7 17 und J774A.1 18 optimiert. Dieser Ansatz nutzt die Amaxa Nucleofector 96-well Shuttle System zur Transfektion; Dieses Systemverwendet eine Kombination von Spezialreagenzien und Elektroporation von Zellen in 96-well Format 19 transfizieren. Nach der Transfektion beschreiben wir Methoden zur Zellgewinnung und Lebensfähigkeit zu maximieren und die anschließende siRNA-induzierte Gen Knockdown zu maximieren. Um den Nutzen dieses Ansatzes zu verdeutlichen, beschreiben wir ein Protokoll für siRNA auf diese Makrophagen-Zelllinien, diese Zellen zu stimulieren mit Lipopolysaccharid (LPS) und überwachen die angeborene Immunantwort auf der Ebene der Produktion von mehreren pro-inflammatorischen Zytokinen. Wir stellen Beispieldaten, in denen wir den Toll-like Rezeptor (TLR) Familie, deren Mitglieder spüren LPS und andere Erreger associated molecular patterns (PAMPs), um angeborene Immunität regulieren Ziel.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Amaxa nucleofector 96-well shuttle system</td>
			<td>Lonza</td>
			<td>AAM-1001S</td>
		<td></td></tr>
		<tr >
			<td >Amaxa SF cell line 96-well nucleofector kit</td>
			<td>Lonza</td>
			<td>V4SC-2096</td>
		<td></td></tr>
		<tr >
			<td >RAW264.7 mouse macrophage cell line</td>
			<td>ATCC</td>
			<td>TIB-71</td>
		<td></td></tr>
		<tr >
			<td >J774A.1 mouse macrophage cell line</td>
			<td>ATCC</td>
			<td>TIB-67</td>
		<td></td></tr>
		<tr >
			<td >siGenome smartpool siRNA</td>
			<td>Dharmacon</td>
			<td>varies depending on gene</td>
		<td></td></tr>
		<tr >
			<td >Non-targeting control siRNA pool</td>
			<td>Dharmacon</td>
			<td>D-001206-13-20</td>
		<td></td></tr>
		<tr >
			<td >Block-iT fluorescent oligo for electrooration</td>
			<td>Invitrogen</td>
			<td>13750062</td>
		<td></td></tr>
		<tr >
			<td >Ultrapure E. coli O111:B4 LPS</td>
			<td>List Biological Laboratories</td>
			<td>421</td>
		<td></td></tr>
		<tr >
			<td >DMEM, high glucose</td>
			<td>Invitrogen</td>
			<td>11995-065</td>
		<td></td></tr>
		<tr >
			<td >RPM1-1640</td>
			<td>Invitrogen</td>
			<td>11875-093</td>
		<td></td></tr>
		<tr >
			<td >Penicillin-Streptomycin Solution (Pen/Strep)</td>
			<td>Fisher</td>
			<td>SV30010</td>
		<td></td></tr>
		<tr >
			<td >0.25% Trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25200072</td>
		<td></td></tr>
		<tr >
			<td >96 well tissue culture plates</td>
			<td>Fisher</td>
			<td>07-200-89 </td>
		<td></td></tr>
		<tr >
			<td >96 well round bottom sterile plates (not coated)</td>
			<td>Fisher</td>
			<td>07-200-745</td>
		<td></td></tr>
		<tr >
			<td >Mouse IL-6 Duoset ELISA kit</td>
			<td>R&#38;D Biosystems</td>
			<td>DY406</td>
		<td></td></tr>
		<tr >
			<td >Mouse TNFa Duoset ELISA kit</td>
			<td>R&#38;D Biosystems</td>
			<td>DY410</td>
		<td></td></tr>
		<tr >
			<td >Fluorescein diacetate</td>
			<td>Sigma-Aldrich</td>
			<td>F7378</td>
		<td></td></tr>
		<tr >
			<td >RLT Bufer</td>
			<td>Qiagen</td>
			<td>79216</td>
		<td></td></tr>
		<tr >
			<td >Rneasy mini kit</td>
			<td>Qiagen</td>
			<td>74134</td>
		<td></td></tr>
		<tr >
			<td >Vybrant Phagocytosis Assay Kit</td>
			<td>Invitrogen</td>
			<td>V-6694</td>
		<td></td></tr>
	</tbody>
</table>

using rna-interference to investigate the innate immune response in mouse macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

Um die Effizienz der Transfektion unter Verwendung dieses Ansatzes zu demonstrieren, verfolgten wir die Aufnahme von FITC-markierter siRNA unter Verwendung eines Durchflusszytometers (Abbildung 1). Um die Brauchbarkeit des Ansatzes zur Überwachung der angeborenen Immunantwort zu veranschaulichen, transfizierten wir siRNAs bekannten angeborenen Immunregulationsgene in die RAW264.7 Makrophagen-Zelllinie, die Zellen stimuliert mit LPS und dann überwacht Produktion der proinfl...

Watch this Scientific Journal Video about Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages at JoVE.com

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Mit RNA-Interferenz, um die angeborene Immunantwort in Makrophagen der Maus Untersuchen

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

using-rna-interference-to-investigate-innate-immune-response-mouse

Research

JoVE Journal

Immunology and Infection

10.6K Aufrufe.  National Jewish Health and University of Colorado School of Medicine. In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Serie: Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

RNA Interference in Ticks

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors of human diseases1 and the most important vectors affecting cattle industry worldwide2. Ticks are classified in the subclass Acari, order Parasitiformes, suborder Ixodida and are distributed worldwide from Arctic to tropical regions3. Despite efforts to control tick infestations, these ectoparasites remain a serious problem for human and animal health4,5.
RNA interference (RNAi)6 is a nucleic acid-based reverse genetic approach that involves disruption of gene expression in order to determine gene function or its effect on a metabolic pathway. Small interfering RNAs (siRNAs) are the effector molecules of the RNAi pathway that is initiated by double-stranded RNA (dsRNA) and results in a potent sequence-specific degradation of cytoplasmic mRNAs containing the same sequence as the dsRNA trigger7-9. Post-transcriptional gene silencing mechanisms initiated by dsRNA have been discovered in all eukaryotes studied thus far, and RNAi has been rapidly developed in a variety of organisms as a tool for functional genomics studies and other applications10.
RNAi has become the most widely used gene-silencing technique in ticks and other organisms where alternative approaches for genetic manipulation are not available or are unreliable5,11. The genetic characterization of ticks has been limited until the recent application of RNAi12,13. In the short time that RNAi has been available, it has proved to be a valuable tool for studying tick gene function, the characterization of the tick-pathogen interface and the screening and characterization of tick protective antigens14. Herein, a method for RNAi through injection of dsRNA into unfed ticks is described. It is likely that the knowledge gained from this experimental approach will contribute markedly to the understanding of basic biological systems and the development of vaccines to control tick infestations and prevent transmission of tick-borne pathogens15-19.

A method for RNA interference (RNAi) by injection of dsRNA into unfed ticks is described. RNAi is the most widely used gene-silencing technique in ticks where the use of other methods of genetic manipulation has been limited.

RNA-Interferenz in Ticks

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors ...

Forschung

Immunologie und Infektion

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

Eine Einführung in die parasitischen Wespen<em&gt; Drosophila</em&gt; Und die antiparasitäre Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

Intravital Imaging of the Mouse Thymus mittels 2-Photonen-Mikroskopie

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

Quantitative Messung der Immunantwort und Sleep in<em&gt; Drosophila</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

The phagocyte respiratory burst is part of the innate immune response to pathogen infection and involves the production of reactive oxygen species (ROS). ROS are toxic and function to kill phagocytized microorganisms. In vivo quantification of phagocyte-derived ROS provides information regarding an organism's ability to mount a robust innate immune response. Here we describe a protocol to quantify and compare ROS in whole zebrafish embryos upon chemical induction of the phagocyte respiratory burst. This method makes use of a non-fluorescent compound that becomes fluorescent upon oxidation by ROS. Individual zebrafish embryos are pipetted into the wells of a microplate and incubated in this fluorogenic substrate with or without a chemical inducer of the respiratory burst. Fluorescence in each well is quantified at desired time points using a microplate reader. Fluorescence readings are adjusted to eliminate background fluorescence and then compared using an unpaired t-test. This method allows for comparison of the respiratory burst potential of zebrafish embryos at different developmental stages and in response to experimental manipulations such as protein knockdown, overexpression, or treatment with pharmacological agents. This method can also be used to monitor the respiratory burst response in whole dissected kidneys or cell preparations from kidneys of adult zebrafish and some other fish species. We believe that the relative simplicity and adaptability of this protocol will complement existing protocols and will be of interest to researchers who seek to better understand the innate immune response.

The innate immune response protects organisms against pathogen infection. A critical component of the innate immune response, the phagocyte respiratory burst, generates reactive oxygen species that kill invading microorganisms. We describe a respiratory burst assay that quantifies reactive oxygen species produced when the innate immune response is chemically induced.

Die Quantifizierung der Atem Burst Antwort als Indikator für Angeborene Immunsystem Gesundheit in Zebrafisch

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine &#947;HV68 is a natural pathogen of murine rodents. &#947;HV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that &#947;HV68 hijacks MAVS and IKK&#946;, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NF&#922;B activation and antiviral cytokine production. Specifically, &#947;HV68 infection activates IKK&#946; and that activated IKK&#946; phosphorylates RelA to accelerate RelA degradation. As such, &#947;HV68 efficiently uncouples NF&#922;B activation from its upstream activated IKK&#946;, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.

We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (&#947;HV68) that is closely-related to human Kaposi&#8217;s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. &#8220;Reconstituting&#8221; the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production.

Zergliedern Angeborene Immunsystem Signaling in Viral Evasion von Zytokin-Produktion

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

In-vitro-Analyse Myd88-vermittelte zelluläre Immunantwort gegen das West-Nil-Virus-Infektion Mutantenstamm

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

Nicht-invasive Bildgebung von der angeborenen Immunantwort in einem Zebrafisch-Larven Modell<em&gt; Streptococcus iniae</em&gt; Infektions

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Using Phylogenetic Analysis to Investigate Eukaryotic Gene Origin

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct a tree to show the evolutionary relationship among different taxa (classification units) at the molecular level. Phylogenetic analysis can also be used to investigate domain relationships within an individual taxon, particularly for organisms that have undergone substantial change in morphology and physiology, but for which researchers lack fossil evidence due to the organisms&#39; long evolutionary history or scarcity of fossilization.
In this text, a detailed protocol is described for using the phylogenetic method, including amino acid sequence alignment using Clustal Omega, and subsequent phylogenetic tree construction using both Maximum Likelihood (ML) of Molecular Evolutionary Genetics Analysis (MEGA) and Bayesian Inference via MrBayes. To investigate the origin of eukaryotic Sugars Will Eventually be Exported Transporters (SWEET) genes, 228 SWEETs including 35 SWEET proteins from unicellular eukaryotes and 57 SemiSWEET proteins from prokaryotes were analyzed. Interestingly, SemiSWEETs were found in prokaryotes, but SWEETs were found in eukaryotes. Two phylogenetic trees constructed using theoretically distinct methods have consistently&#160;suggested that the first eukaryotic SWEET gene might stem from the fusion of a bacterial SemiSWEET gene and an archaeal SemiSWEET gene.&#160;It is worth noting that one should be cautious to draw a conclusion based only on phylogenetic analysis, although it is useful to explain the underlying relationship between different taxa, which is difficult or even impossible to discern through experimental means.

A method of constructing a phylogenetic tree based on sequence homology of SWEETs from eukaryotes and SemiSWEETs from prokaryotes is described. Phylogenetic analysis is a useful tool for explaining the evolutionary relatedness between homologous proteins or genes from different organism groups.

Mit der phylogenetischen Analyse zu eukaryotischen Gens Herkunft untersuchen

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

Zwei-Photon intravitalen Bildgebung der Leukozyten im Rahmen der Immunantwort in Lipopolysaccharid behandelt Maus Leber

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...