Funding from the Kansas Agricultural Experiment Station and K-State Arthropod Genomics Center to KYZ supported the original development of chitosan/dsRNA-mediated gene silencing in A. gambiae19.The A. gambiae work described here was supported in part by R01-AI095842 from NIH/NIAID to KM. Development of chitosan/siRNA-mediated gene silencing in A. aegypti20,21 was supported by NIH/NIAID Award R01-AI081795 to MDS. The contents of this study are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

1. Mückenarten und Aufzucht <ol><li> Pflegen A. gambiae G3 und A. aegypti Liverpool IB12 Stämme (in den repräsentativen Studien unten verwendet) oder andere Stämme von Interesse nach Standardlaborpraxis oder wie zuvor beschrieben 23,24. </li></ol> 2. dsRNA und siRNA-Design und Produktion <ol><li> Design Primer an die dsRNA spezifisch für das Gen von Interesse Vorlagen zu konstruieren. Verwenden Sie die E-RNAi-Tool 25. Produzieren dsRNA gemß der Methode der Wahl oder, wie zuvor beschrieben 19. <ol><li> Für eine negative Kontrolle, synthetisieren dsRNA 19 entsprechend der Sequenz des grün fluoreszierenden Proteins (GFP), β-Galactosidase (β-gal) oder ein anderes Gen nicht in Moskitos ausgedrückt. Falls gewünscht, kann dsRNA dazu verwendet, die unten zusammengefaßt sind repräsentative Ergebnisse erzeugen 19 als eine positive Kontrolle verwendet werden. Shop dsRNA in RNase-freiem Wasser gelöst bei -80 °; C, bis sie benötigt. </li></ol></li><li> Alternativ Design-Gen-spezifische siRNA nach Standardlaborpraxis oder wie zuvor 10 beschrieben. Verwürfeln die Sequenz eines Zuschlags siRNA negative Kontroll-siRNA, die keinem Moskito Gen nicht entspricht entwerfen. Kaufen Sie die kundenspezifischen siRNAs, die durch eine Reihe von seriösen Anbietern zur Verfügung stehen. <ol><li> Falls gewünscht, können siRNAs verwendet, die im Folgenden zusammengefasst sind repräsentative Ergebnisse erhalten 20,21 als positive Kontrollen verwendet werden. Shop siRNA in RNase-freiem Wasser gelöst bei -80 ° C, bis sie benötigt. </li></ol></li></ol> 3. Vorbereitung Chitosan / interfering RNA Nanopartikel <ol><li> Sammeln vorgefertigten RT 100 mM Natriumsulfat (100 mM Na 2 SO 4 in deionisiertem H 2 O) und 0,1 M Natriumacetat (0,1M NaC 2 H 3 O 2 -0.1 M Essigsäure, pH 4,5, in entionisiertem H 2 O) Puffer. </li><li> Auflösen von Chitosan (≥75%deacetylierten) in 0,1 M Natriumacetat-Puffer auf 0,02% (w / v) Arbeitslösung und Halten der Lösung bei RT vor der Verwendung. </li><li> Löse dsRNA oder siRNA in 50 ul entionisiertem H 2 O und sie dem 100 mM Natriumsulfat-Puffer zu einer 100 ul Lösung von 32 ug dsRNA / siRNA pro 100 ul 50 mM Natriumsulfat bilden. </li><li> 100 l Chitosanlösung zur dsRNA / siRNA-Lösung und dann das Gemisch in einem Wasserbad bei 55 ° C für 1 min. Eine Steuer durch Zugabe von 100 ul 50 mM Natriumsulfat zu 100 ul Chitosan-Lösung zu stellen und die gleiche Vorgehensweise. </li><li> Mischen der Lösungen sofort für 30 sec mittels Hochgeschwindigkeits Verwirbeln bei RT, um die Bildung von Nanopartikeln zu erleichtern. </li><li> Zentrifugiere das Gemisch bei 13.000 × g für 10 min bei RT, nach welcher Zeit sich ein Pellet sichtbar sein soll. Den Überstand in ein neues 1,5-ml-Tube. Der Luft trocknen das Pellet für ≈10 Minuten bei Raumtemperatur, bevor Sie es an Moskito Essen zuzubereiten. </li><li> Measure die Konzentration von dsRNA / siRNA im Überstand unter Verwendung des Überstands von der Steuerung (siehe 3.5) als leer, um die Gesamtmenge an dsRNA / siRNA, die im Überstand blieb berechnen. Verwenden der Differenz zwischen den Ausgangsmengen an dsRNA / siRNA und in den Überständen verbleibende, den Prozentsatz der dsRNA / siRNA in Nanopartikel eingeschlossen berechnen Mengen. Dieser Ladeeffizienz ist in der Regel über 90%. </li><li> Wiederholen Sie den Vorgang, wenn mehr Nanopartikel benötigt werden. Verwenden Sie die getrockneten Nanopartikel sofort. Der Einfluss der Kühllagerung der Teilchen vor der Verwendung wurde nicht untersucht. </li></ol> 4. Vorbereitung Mosquito Lebensmittel enthalten Chitosan / interfering RNA Nanopartikel <ol><li> Herstellung von 1 ml einer 2% igen Lösung (w / v) in entionisiertem Wasser, schmelzen die Agarose und Halten geschmolzenen Agarose-Lösung in einem 55 C-Wasserbad vor dem Gebrauch. </li><li> Mix Fischfutter Flocken (47% Rohprotein, min. Rohfett 10%, max. Rohfaser 3%) und Trockenhefe in eineVerhältnis von 2: 1 (w / w). Schleifen Sie die Mischung, um kleine Partikel mit einem Mörser und Stößel (passable bis Nr 50 USA Standard-Test-Sieb). Verwenden Sie die Grundnahrung, die bräunlich gefärbt sein sollte, entweder sofort oder lagern Sie es in einem verschlossenen Behälter bei 4 ° C für mehrere Wochen. </li><li> In einer 1,5-ml-Tube, mischen 6 mg Boden Nahrung mit den getrockneten Nanopartikel aus Abschnitt 3.7 mit einem Zahnstocher. </li><li> Zugabe von 30 ul 2% vorge geschmolzener Agarose-Gel-Lösung auf die Lebensmittelnanopartikelmischung; sofort und gründlich zu rühren, indem Sie einen Zahnstocher oder Pipettenspitze. </li><li> Verwenden Sie das Gel Essen und Nanopartikel enthalten, um die Mückenlarven sofort füttern. Alternativ, wenn das Gel vollständig bei RT erstarrte, speichern das Gel bei 4 ° C lagern und am nächsten Tag oder bei -80 ° C für die spätere Verwendung. </li></ol> 5. Ernährung Mückenlarven mit Lebensmittel, die Chitosan / interfering RNA Nanopartikel <ol><li> Feeding A. gambiae Mückenlarven: <ol><li> Entfernen eines single Gel-Pellet aus der 1,5-ml-Röhrchen mit einem Zahnstocher und schneiden Sie es in 6 gleiche Scheiben mit einem sauberen Rasierklinge oder Zahnstocher. </li><li> Übertragungs 20 dritte Larvenstadium in einen 500 ml Petrischale mit 100 ml entionisiertem Wasser. </li><li> Führen Sie das Mückenlarven, indem eine Scheibe des Gels Pellet (fein in kleine Stücke gehackt) pro Petrischale einmal täglich für vier Tage. Achten Sie darauf, Larven auf dem Pellet, die in der Größe deutlich reduziert werden sollte oder am nächsten Tag völlig abwesend zu beobachten. Nach der Zeit wird Mücken in späten vierten Larvenstadium entwickeln. </li><li> Notieren Sie alle sichtbaren phänotypischen Veränderungen während des Experiments. Untersuchen Sie die Transkriptionsniveaus und andere phänotypische Veränderungen, wie in Abschnitt 6 am Ende der viertägigen Zeitraum diskutiert. </li></ol></li><li> Feeding A. aegypti Mückenlarven: <ol><li> Schneiden Sie die Gel-Pellet in 6 gleiche Scheiben mit einem sauberen Rasierklinge oder Zahnstocher. </li><li> Zeigen 50 alters synchronisiert 24 Stunden nach dem Schlüpfen Ei fierste Larvenstadium in eine Petrischale in ≈40 ml entionisiertem Wasser. </li><li> Feed-Larven eine Scheibe pro Petrischale 4 Stunden / Tag, dann Transfer Larven zurück zu den regelmäßigen Larven Ernährung von 2: 1 Bodenfischfutter Flocken und Trockenhefe für den Rest des Tages. Wiederholen Sie den Vorgang täglich während der vier Larvenstadien. </li><li> Untersuchen Sie die Transkriptionsniveaus und andere phänotypische Veränderungen, wie in Abschnitt 6 an den gewünschten Entwicklungszeitpunkten diskutiert. </li></ol></li></ol> 6. Bestätigung der Gene Knockdown <ol><li> Relative Transkript Quantifizierung durch qRT-PCR in A. aegypti und A. gambiae Larven. <ol><li> Führen und qRT-PCR-Assays zu analysieren mit drei biologischen Replikaten auf mindestens 10 gepoolten Kontroll vs. experimentelle Larven, wie beschrieben, 11,19,20, oder nach Standard-Laborverfahren. Repräsentative Ergebnisse sind unten aufgeführt. </li><li> Für die Analyse eines bestimmten Gewebetyp / Körperteil (dh., Gehirn oder Antenne), perform qRT-PCR, wie in 6.1.1 folgende Präparation beschrieben, um das Gewebe von Interesse zu erholen (siehe zB Mysore et al. 21). </li></ol></li><li> Bestätigung der Zuschlags durch in situ Hybridisierung <ol><li> Synthetisieren Digoxygenin-markierte Antisense-und Sense-Steuer Ribosonden nach Standardlaborpraxis oder wie beschrieben 26. </li><li> Ausführen der in situ Hybridisierung gemäß dem Haugen et al. 27 Protokoll zur Bestätigung der Zuschlags wie zuvor 11,20 beschriebene und in der repräsentative Ergebnisse weiter unten diskutiert. </li></ol></li><li> Bestätigung der Zuschlags immunhistochemisch <ol><li> Wenn Antikörper gegen das Proteinprodukt des Gens gezielt zur Verfügung stehen, ausführen Immunhistochemie wie zuvor beschrieben 28, die Identifizierung von Individuen mit den höchsten Niveaus der Zuschlags für Phänotypanalyse 20 erleichtert, wie in der Vertre exemplifiziertve Ergebnisse unten. </li></ol></li></ol>

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The authors have nothing to disclose.

Das Chitosan / interfering RNA Nanopartikel hierin beschriebenen Methodik wurde verwendet, um Gene während Larvenentwicklung in A. zielgerichteter gambiae (2, 3) und A. aegypti (Figuren 4, 5, 6, Tabelle 1, 2). Chitosan-Nanopartikeln kann entweder mit langen dsRNA oder siRNA, die beide erfolgreich in Moskitos verwendet worden, wie durch die hierin beschriebenen repräsentativen Ergebnisse zeigten weiterhin hergestellt werden. Synthese der dsRNA ist weniger teu...

Blut-Fütterung Trägermücken der Anopheles und Aedine Gattungen übertragen Krankheitserreger für einige der schlimmsten Geißeln der Menschheit verantwortlich. Schätzungsweise 3,4 Milliarden Menschen mit einem Risiko für Malaria zu erkranken, die für mehr als eine halbe Million Todesfälle pro Jahr weltweit verantwortlich ist. Malaria Ergebnisse vor einer Infektion durch Plasmodium sp. Parasiten, die den Menschen durch den Stich infizierter Mücken auf der Gattung Anopheles, einschließlich der Hauptnischen Vektor Anopheles gambiae (übertragen werden <a href="http://www.who.int/topics/malaria/en/">http://www.who.int/topics/malaria/en/</a> , 2014) 1. Aedes aegypti ist die primäre Trägermücke für Dengue-Virus, die Dengue-Fieber, eine unspezifische fieberhafte Erkrankung, die die am weitesten verbreitete und wichtige arboviral Krankheit in der Welt ist, verursacht. Dengue-Virus ist derzeit eine Bedrohung für&gt; 2,5 Milliarden Menschen in den Tropen, mit einer jährlichen incidence von rund 50 Millionen Fällen zu ~ 24.000 Todesfälle pro Jahr ( <a href="http://www.cdc.gov/dengue/">http://www.cdc.gov/dengue/</a> , 2014) 2. Trotz der dramatischen weltweiten Auswirkungen der Moskitos übertragene Krankheiten auf die menschliche Gesundheit sind effiziente Mittel zur Prävention und Behandlung dieser Krankheiten fehlt. Moskito-Kontrolle ist derzeit die beste Methode zur Verhütung von Krankheiten. Das Potenzial für die Steuerung von Arthropoden übertragene Krankheiten, die durch die genetische Manipulation von Wirtsinsekten ist seit mehr als vier Jahrzehnten 3 anerkannt worden. Transgene Stämme von A. aegypti entwickelt, um eine reprimierbaren frauenspezifische flug Phänotyp haben in jüngster Zeit, das Potenzial für den Einsatz von transgenen Kontrollstrategien zu verwirklichen 4-6. Diese Fortschritte haben die Forscher herausgefordert, neue genetische Ziele für Vektorregelung und zusätzliche Mittel zum Manipulieren der Genfunktion in Überträgermücken zu identifizieren. Eine Veränderung der Genexpression dährend Entwicklung, wie in weiblich-flug Mücken 4 der Fall war, kann die Aufklärung von neuen Kontrollstrategien zu fördern. Jedoch hauptsächlich auf technische Herausforderungen, die Funktionen der wenigen Genen während der Entwicklung A. gekennzeichnet gambiae, A. aegypti oder andere Mückenarten. Seit ihrer Entdeckung in C. elegans 7, RNA-Interferenz (RNAi), die in Tieren, Pflanzen und Mikroorganismen konserviert ist, wurde umfassend für funktionelle genetische Untersuchungen in einer Vielzahl von Organismen, einschließlich Insekten 8,9 verwendet. Die RNAi wird von Dicer, das spaltet lange dsRNA in kurze 20-25 Nukleotide langen siRNAs, die als sequenzspezifische interfering RNA funktionieren begonnen. siRNAs Stille Gene, die in der Sequenz komplementär sind durch die Förderung Transkript Umsatz, Spaltung, und die Störung der Übersetzung 9. Lange dsRNA-Moleküle (in der Regel 300 bis 600 bp) oder kundenspezifischen siRNAs eine particular Sequenz kann im Forschungslabor zur Dämpfung jedes Gen von Interesse verwendet werden. Durch die Verwaltung, wenn interfering RNA geliefert wird, können die Forscher die Zeit zu steuern, mit der Gen-Silencing-Eingeweihten. Dieser Vorteil ist nützlich, da es verwendet werden, um Herausforderungen wie die Entwicklung Letalität oder Sterilität, die die Produktion und Wartung von Stämmen Lager vererbbare Mutationen, eine kostspielige und arbeitsintensiver Prozess, der nicht in allen Insektenarten ist noch behindern überwunden werden können. Obwohl der Grad der Gen-Silencing durch RNAi von Gen zu Gen, Gewebe zu Gewebe und Tier zu Tier variieren, wird RNAi weithin für die funktionelle Analyse von Genen in Moskitos und andere Insekten 8,9 verwendet. Drei interfering RNA Lieferstrategien in Mücken verwendet: Mikroinjektion, Einweichen / topische Anwendung und Einnahme. Für eine detaillierte Geschichte und ein Vergleich der Verwendung dieser drei Techniken in Insekten, wenden Sie sich bitte an Yu et al 8 beziehen. WE erfolgreich als Mittel zur Bereitstellung von siRNAs verwendet Mikroinjektion von 10 bis Entwicklungsgenen in A. Ziel aegypti Embryos, Larven und Puppen 11-14. Diese arbeitsintensive Lieferstrategie erfordert jedoch sowohl eine Mikroinjektion Einrichtung und eine geschickte Hand. Darüber hinaus ist die Mikroinjektion stressig für den Organismus, ein Störfaktor, vor allem wenn Verhaltensphänotypen bewertet werden. Schließlich können die Mikroinjektion Lieferung nicht auf den Bereich für die Vektorregelung verlängert werden. Alternativ Einweichen der Organismus in interfering RNA-Lösung hat sich auch ein beliebtes Mittel zur Induktion Gen-Silencing, wie es bequem und erfordert wenig Ausrüstung oder die Arbeitskräfte. Einweichen wurde hauptsächlich in Insektenzelllinie angewendet wurde studiert 8, jedoch in einer neuen Studie wurde Zuschlag in A. erreicht aegypti Larven in einer Lösung von dsRNA 15, die die Tiere zu sein schien Einnahme eingetaucht. Für Studien, die Analyse von mehreren experimental Gruppen oder Phänotypen ist Einweichen ziemlich teuer. Lopez-Martinez et al. 16 beschrieben Rehydrierung angetrieben RNAi, einen neuen Ansatz für interfering RNA Lieferung, die Dehydratisierung in Kochsalzlösung und Rehydrierung beinhaltet mit einem einzigen Tropfen Wasser enthält interfering RNA. Dieser Ansatz bedeutet Senkung der Kosten mit ganzen Tiereint assoziiert, aber teurer ist als die Mikroinjektion und in ihrer Anwendung nicht auf Spezies, die eine hohe osmotische Druck tolerieren kann begrenzt werden. Darüber hinaus ist es schwierig, sich vorzustellen, wie Tauchen oder Dehydratation / Rehydratation Tauch Methode könnte zur Vektor-Kontrolle im Bereich angepasst werden. Aus diesen Gründen ist für die post-embryonale Studien, ist die Lieferung von interfering RNA mit aufgenommenen Nahrung eine Alternative Strategie. Obwohl Einnahme basierte Strategien müssen nicht in allen Insektenarten, vor allem wohl Drosophila melanogaster ist orale Verabreichung von interfering RNA mit der Nahrung vermischt Gen si gefördertlencing in eine Vielzahl von Insekten 8,17, einschließlich A. aegypti Erwachsene 18. Wir beschriebenen Chitosan-Nanopartikel vermittelten RNAi in A. gambiae Larven 19 und haben diesen Ansatz zur Reduktion der Genexpression in A. erfolgreich angewendet aegypti Larven 20,21. Hier Methodik für diese RNAi Verfahren, das Einschließen von interfering RNA durch das Polymer Chitosan umfasst, ist detailliert. Chitosan / interfering RNA Nanopartikel durch Selbstorganisation von Polykationen mit interferierenden RNA durch die elektrostatischen Kräfte zwischen den positiven Ladungen der Aminogruppen in Chitosan und negativen Ladungen von den Phosphatgruppen auf der Hauptkette des interferierenden RNA 19 durch gebildet. Das beschriebene Verfahren ist sowohl mit langen dsRNA-Moleküle (im folgenden als dsRNA bezeichnet) oder doppelsträngigen siRNA (nachstehend als siRNA bezeichnet) kompatibel. Nach der Synthese Chitosan / interfering RNA Nanopartikel mit Futtersaft gemischt und geliefertLarven bei Einnahme. Diese Methode ist relativ kostengünstig, erfordert wenig Ausrüstung und Arbeits 19 und ermöglicht Hochdurchsatz-Analyse von mehreren Phänotypen, einschließlich der Analyse der Verhaltensweisen 20,21. Diese Methodik, die für die Gen-Silencing-Studien in anderen Insekten, einschließlich anderer Krankheitsvektoren und Insekten landwirtschaftlichen Schädlingen angepasst werden kann, könnte möglicherweise für das Gen-Silencing in einer Vielzahl von anderen Tierspezies verwendet werden. Außerdem könnte Chitosan, ein kostengünstiges, nicht-toxische und biologisch abbaubares Polymer 22, möglicherweise in dem Feld für artspezifischen Moskitobekämpfung genutzt werden.

<table border="0" cellpadding="0" cellspacing="0" width="1429">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Name of Reagent/Equipment</td>
			<td style="width:243px;">Company</td>
			<td style="width:177px;">Catalog Number</td>
			<td style="width:663px;">Comments/Descriptions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.02 ml pipetteman</td>
			<td>Rainin</td>
			<td>PR20</td>
			<td>for resuspension of interfering RNA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.2 ml pipetteman</td>
			<td>Rainin</td>
			<td>PR200</td>
			<td>for resuspension of interfering RNA and preparation of interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.5 ml graduated tube&#160;</td>
			<td>Fischer Scientific</td>
			<td>05-408-120</td>
			<td>for aliquoting resuspended interfering RNA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 ml pipetteman</td>
			<td>Rainin</td>
			<td>PR1000</td>
			<td>for resuspension of interfering RNA and preparation of interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.5 ml graduated tube&#160;</td>
			<td>Fischer Scientific</td>
			<td>05-408-046</td>
			<td>for preparation of interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1M Sodium Acetate, pH 4.5</td>
			<td>TEKnova</td>
			<td>S0299</td>
			<td>to prepare sodium acetate buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetic Acid, AIRSTAR. ACS, USP, FCC Grade</td>
			<td>BDH</td>
			<td>VWR BDH3092-500MLP</td>
			<td>to prepare sodium acetate buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agarose Genetic Technology Grade</td>
			<td>MP Biomedicals LLC.</td>
			<td>800668</td>
			<td>to coat prepared interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge</td>
			<td>Eppendorf AG</td>
			<td>5415D</td>
			<td>to pellet interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chitosan, from shrimp shells</td>
			<td>Sigma-Aldrich</td>
			<td>C3646-25G</td>
			<td>to combine with interfering RNA for prepararation of interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dry Yeast</td>
			<td>Universal Food Corp</td>
			<td>NA</td>
			<td>to prepare mosquito larval food with interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">E-RNAi tool</td>
			<td>German Cancer Research Center</td>
			<td>NA</td>
			<td>for design of dsRNA; http://www.dkfz.de/signaling/e-rnai3//</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">goldfish food</td>
			<td>Wardley</td>
			<td>Goldfish FLAKE FOOD</td>
			<td>to prepare mosquito larval food with interfering RNA/nanoparticles</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Heated water bath</td>
			<td>Thermo Scientific</td>
			<td>51221048</td>
			<td>to heat the interfering RNA/nanoparticles at 55oC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ice</td>
			<td>not applicable</td>
			<td>not applicable</td>
			<td>for thawing interfering RNA and preparation of interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ice bucket</td>
			<td>Fisher Scientific</td>
			<td>02-591-44</td>
			<td>for storage of ice used during the procedure</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">liver powder</td>
			<td>MP Biomedicals LLC.</td>
			<td>900396</td>
			<td>to feed mosquito larvae post interfering RNA/nanoparticle treatment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microwave oven</td>
			<td>A variety of vendors</td>
			<td>not applicable</td>
			<td>to prepare 2% agarose solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">petridish (100X15 mm)</td>
			<td>Fischer Scientific</td>
			<td>875713</td>
			<td>interfering RNA/nanoparticle feeding chamber for larvae</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pH meter</td>
			<td>Mettler Toledo</td>
			<td>S220</td>
			<td>for preparation of buffers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Razor blade</td>
			<td>Fischer Scientific</td>
			<td>12-640</td>
			<td>to divide the interfering RNA/nanoparticle pellet for feedings</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">siRNA</td>
			<td>Thermo Scientific/Dharmacon</td>
			<td>custom</td>
			<td>for preparation of siRNA/nanoparticles</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Sodium Sulfate, Anhydrous (Na2SO4)</td>
			<td>BDH/distributed by VWR</td>
			<td>VWR BDH0302-500G</td>
			<td>to prepare 50mM sodium sulfate solution in which the interfering RNA will be resuspended</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thermometer</td>
			<td>VWR</td>
			<td>61066-046</td>
			<td>to measure the water bath temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tooth picks</td>
			<td>VWR</td>
			<td>470146-908</td>
			<td>for stirring during interfering RNA/nanoparticle food preparation and cutting gel pellets</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Ultralow freezer</td>
			<td>A variety of vendors</td>
			<td>not applicable</td>
			<td>for storage of interfering RNA aliquots and interfering RNA/nanoparticles at -80oC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vortex mixer</td>
			<td>Fischer Scientific</td>
			<td>02-216-108</td>
			<td>for preparation of chitosan/interfering RNA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Weight paper</td>
			<td>Fischer Scientific</td>
			<td>NC9798735</td>
			<td>to divide the interfering RNA/nanoparticle pellet for feedings</td>
		</tr>
	</tbody>
</table>

chitosan/interfering rna nanoparticle mediated gene silencing in disease vector mosquito larvae

Vektor-Mücken verursachen mehr menschliches Leid als jeder andere Organismus - und tötet mehr als eine Million Menschen pro Jahr. Die Moskitogenomprojekten erleichtert die Forschung in neue Facetten Moskito Biologie, einschließlich funktionale genetische Studien in der Primär afrikanischen Malariavektor Anopheles gambiae und dem Dengue-Fieber und Gelbfieber-Vektor Aedes aegypti. RNA Interferenz- (RNAi-) vermittelte Gen-Silencing ist verwendet worden, um Gene von Interesse in beiden dieser Krankheitsvektor Mückenarten zielen. Hier beschreiben wir ein Verfahren zur Herstellung von Chitosan / interferierenden RNA-Nanopartikel, die mit Lebensmitteln kombiniert und durch Larven aufgenommen. Diese technisch einfach, mit hohem Durchsatz und relativ kostengünstige Methode, die mit langen doppelsträngigen RNA (dsRNA) oder kleine interferierende RNA (siRNA) Moleküle kompatibel ist, ist für die erfolgreiche Knockdown von einer Anzahl von verschiedenen Genen in A. verwendet gambiae und A. aegyptiLarven. Nach Larvenernährung, Knockdown, die durch qRT-PCR und in situ Hybridisierung überprüft wird, kann zumindest durch die späten Puppenstadium fortbestehen. Diese Methode kann für eine Vielzahl von vor allem Insektenarten, darunter landwirtschaftliche Schädlinge sowie andere nicht-Modellorganismen sein. Zusätzlich zu ihrer Nützlichkeit im Forschungslabor, in der Zukunft, Chitosan, einem kostengünstigen, nicht-toxischen und biologisch abbaubaren Polymer, könnte möglicherweise in dem Gebiet verwendet werden.

<html> A. gambiae: Chitosan / dsRNA Nanopartikel aufgrund der elektrostatischen Wechselwirkung zwischen den Aminogruppen des Chitosans und der Phosphatgruppen des dsRNA gebildet. Die Effizienz der dsRNA Einbau in Nanopartikel der Regel über 90%, wie durch Abreicherung von dsRNA aus der Lösung gemessen. Rasterkraftmikroskopische Aufnahmen zeigen, dass Chitosan-dsRNA Partikelgröße im Durchschnitt 140 nm im Durchmesser reichen von 100 bis 200 nm (Abbildung 1...

Watch this Scientific Journal Video about Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae at JoVE.com

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Hier beschreiben wir ein Verfahren zur Hemmung der Genfunktion in Krankheitsvektor Mücken durch die Verwendung von Chitosan / interferierenden RNA-Nanopartikel, die durch Larven aufgenommen werden.

Chitosan / interfering RNA Nanopartikel vermittelten Gen-Stummschaltung in Disease Vector Mückenlarven

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects ...

chitosaninterfering-rna-nanoparticle-mediated-gene-silencing-disease

Research

JoVE Journal

Biology

14.2K Aufrufe.  Kansas State University.  Hier beschreiben wir ein Verfahren zur Hemmung der Genfunktion in Krankheitsvektor Mücken durch die Verwendung von Chitosan / interferierenden RNA-Nanopartikel, die durch Larven aufgenommen werden. 

Serie: Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

Virus-induzierten Gene Silencing (VIGS) in Nicotiana benthamiana Und Tomaten

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

Forschung

Biologie

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

Live Cell Imaging mit Calcium siRNA vermittelte Gene Silencing Combined Identifiziert Ca 2 + Leck-Kanäle in der ER-Membran und deren Regulationsmechanismen

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

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 ...

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 ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Mit SECM Arrest Sequenz als Werkzeug zum Ribosom gebunden Polypeptide isolieren

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity and functionality of the gut community will provide insight into the effects of gut microbiota on mosquito life traits. Metagenomic RNA-Seq has become an important tool to analyze transcriptomes from various microbes present in a microbial community. Messenger RNA usually comprises only 1-3% of total RNA, while rRNA constitutes approximately 90%. It is challenging to enrich messenger RNA from a metagenomic microbial RNA sample because most prokaryotic mRNA species lack stable poly(A) tails. This prevents oligo d(T) mediated mRNA isolation. Here, we describe a protocol that employs sample derived rRNA capture probes to remove rRNA from a metagenomic total RNA sample. To begin, both mosquito and microbial small and large subunit rRNA fragments are amplified from a metagenomic community DNA sample. Then, the community specific biotinylated antisense ribosomal RNA probes are synthesized in vitro using T7 RNA polymerase. The biotinylated rRNA probes are hybridized to the total RNA. The hybrids are captured by streptavidin-coated beads and removed from the total RNA. This subtraction-based protocol efficiently removes both mosquito and microbial rRNA from the total RNA sample. The mRNA enriched sample is further processed for RNA amplification and RNA-Seq.

A ribosomal RNA (rRNA) depletion protocol was developed to enrich messenger RNA (mRNA) for RNA-seq of the mosquito gut metatranscriptome. Sample specific rRNA probes, which were used to remove rRNA via subtraction, were created from the mosquito and its gut microbes. Performance of the protocol can result in the removal of approximately 90-99% of rRNA.

Depletion der ribosomalen RNA für Mosquito Gut metagenomische RNA-seq

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity ...

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling1,2, while LRRK2 is implicated in the pathogenesis of Parkinson's disease3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with 32P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with 32P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.

Metabolische Markierung von Leucin Rich-Repeat-Kinasen 1 und 2 mit radioaktivem Phosphat

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, ...

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 ...

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

Meiotischen Spindel Assessment in Maus-Oozyten von siRNA-vermittelte Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and diseases, such as obesity and fatty liver. Usually, lipids are stored in lipid droplets, which are the multifunctional lipid storage organelles in cells. Lipid droplets vary in size and number in different tissues and under different conditions. It has been reported that lipid droplets are tightly controlled through regulation of its biogenesis and degradation. In Drosophila melanogaster, the oenocyte is an important tissue for lipid metabolism and has been recently identified as a human liver analogue regarding lipid mobilization in response to stress. However, the mechanisms underlying the regulation of lipid droplet metabolism in oenocytes remain elusive. To solve this problem, it is of utmost importance to develop a reliable and sensitive method to directly visualize lipid droplet dynamic changes in oenocytes during development and under stressful conditions. Taking advantage of the lipophilic BODIPY 493/503, a lipid droplet-specific fluorescent dye, described here is a detailed protocol for the dissection and subsequent lipid droplet staining in the oenocytes of Drosophila larvae in response to starvation. This allows for qualitative analysis of lipid droplet dynamics under various conditions by confocal microscopy. Furthermore, this rapid and highly reproducible method can also be used in genetic screens for indentifying novel genetic factors involving lipid droplet metabolism in oenocytes and other tissues.

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Presented here are detailed methods for the dissection and lipid droplet staining of oenocytes in Drosophila larvae using BODIPY 493/503, a lipid droplet-specific fluorescent dye.

Dissektion und Lipid Tröpfchen Färbung von Oenocytes in Drosophila Larven

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and ...