Arbeit wurde durch Grant nicht unterstützt. 01GU0615 des &quot;Bundesministerium für Forschung und Technologie (BMBF).

1) Bakterielle Lieferung von shRNAs <ol><li> Bevor Sie mit der Bakterienkultur, muss man LB-Medium und LB-Agar vorzubereiten. </li><li> Für die LB-Medium abwiegen Hefeextrakt (0,5% w / v), Bacto Trypton (1,0% w / v) und NaCl (0,6% w / v) gelöst und diese Komponenten in aqua bidest. Die vorbereiteten Lösungen müssen in einem Autoklaven sterilisiert werden und sind dann einsatzbereit. </li><li> Für LB-Agarplatten Bacto-Agar (1,5% w / v) muss an die LB-Medium früheren zur Sterilisation hinzugefügt werden. </li><li> Aufheizen LB-Agar in der Mikrowelle bis zur LB-Agar vollständig gelöst ist. Lassen Sie die Lösung abkühlen, bis die Flasche leicht und ohne sich zu verbrennen berührt werden. Add Kanamycin (100 mg / ml) für die Auswahl der positiven Klone in weiteren Schritten. </li><li> Bereiten LB-Agar-Platten bei einer laminaren Strömung Bank (sterile Bedingungen) durch Pipettieren 20 ml des LB-Agar-Lösung in 10-cm Petrischalen. Lassen Sie die Platten abkühlen, bis die Flüssigkeit fest geworden ist. Die Platten sind jetzt einsatzbereit und kann bei 4 ° C gelagert werden. </li><li> ShRNA-kodierende Expressionsvektoren sind in kompetente E. verwandelt coli ceq221 durch Hitzeschock mit der CaCl 2-Verfahren. </li><li> Platte der transformierten Bakterien auf LB-Agarplatten mit 100 pg / ml Kanamycin, den Deckel schließen, verschließen Sie die Platten mit Parafilm und pflegen den Kopf bei 37 ° C über Nacht. </li><li> Impfen bakterielle mini Kulturen mit positiven Klone durch Abnehmen gewachsenen Kolonien mit einem Zahnstocher. Übertragen Sie die Zähne in 7,0 ml frisches LB-Medium mit 100 mg / ml Kanamycin und pflegen bei 37 ° C über Nacht auf einem Schüttler bei 200 Umdrehungen pro Minute zu pflücken. </li><li> Inoculate 100 ml frischem LB-Medium mit 100 mg / ml Kanamycin 1:100 mit dem über Nacht Mini-Kultur in einem 1 l-Erlenmeyerkolben, und bei 37 ° C über Nacht auf einem Schüttler bei 200 Umdrehungen pro Minute. </li><li> Seed 25 x 10 4; menschlichen Magen-Karzinom-Zellen (EPG85-257RDB) pro Well in 6 (Anzahl der ausgesäten Zellen hängt von der Geschwindigkeit des Zellwachstums der nach Zelllinie Konfluenz sollten ~ 70-80% 24 Stunden nach der Aussaat werden) - auch Speisen und inkubieren diese bei 37 ° C, in einem 5% CO 2, Wasserdampf gesättigten Atmosphäre über Nacht. </li><li> Zurück zur Krebszelle Infektion über Nacht Kulturen tk RNAi-Vektor-haltigen E. coli sind in einem Photometer die OD 600 zu bestimmen. Verdünnen Sie die bakterielle Lösung bis eine optische Dichte von 0,5 erreicht ist (OD 600 = 0,5 gleich 1,6 x 10 8 Bakterienzellen / ml). </li><li> Ersetzen Sie die FCS-haltigen Zellkulturmedium der Karzinom-Zellen gegen FCS-freiem Medium 30 min früheren bakterielle Co-Inkubation. </li><li> Wash Bakterien zweimal mit 1 x PBS, und verdünnen in serum-freien Leibovitz L-15-Medium. </li><li> Add verdünnt Bakterien, Krebszellen in der gewünschten MOI (Multiplizität der Infektion = Anzahl der Bakterienzellen pro Krebszelle) und Co-Inkubation von Bakterien und Krebszellen für 2 Stunden bei 37 ° C. </li><li> Nach 2 Stunden Co-Inkubation Krebszellen wurden zweimal mit 1x x PBS gewaschen und einmal mit serumhaltigem Leibovitz L-15-Medium mit 100 U / ml Penicillin, 100 ug / ml Streptomycin, 2,5 pg / ml Amphotericin, 150 ug / ergänzt ml Gentamicin und 100 ug / ml Kanamycin. </li><li> Weiter pflegen die Krebszellen bei 37 ° C in einer 5% CO 2, Wasserdampf gesättigten Atmosphäre. </li><li> Die Auswirkungen der bakteriellen Behandlung kann durch Fluoreszenz-Mikroskopie, durch quantitative real-time RT-PCR auf mRNA-Ebene gemessen und mittels Western-Blot-Analyse auf Protein-Ebene sichtbar gemacht und gezeigt, mit funktionellen Assays wie FACS-Analyse und Zellproliferationsassays. (Falls gewünscht, können diese Verfahren im Detail beschrieben werden). </li></ol> 2) repräsentative Ergebnisse Wenn das Protokoll korrekt durchgeführt wird, sollten die Ergebnisse vergleichbar sein, die nachfolgend dargestellt ist. Fluoreszenzmikroskopie Abbildung 1 zeigt einen menschlichen Magenkarzinom Zelle drei Stunden nach der bakteriellen Behandlung (a) im Vergleich zu einer unbehandelten Zellen (b). Rund um den Kern der behandelten Zelle, können Bakterien nachgewiesen werden. <img src="/files/ftp_upload/2099/2099fig1.jpg" alt="Abbildung 1" /> Abbildung 1: Fluoreszenz-Mikroskopie menschlichen Magenkarzinom Zelle nach der bakteriellen Behandlung (1:500) und einer unbehandelten menschlichen Magenkarzinom Zelle als Steuer-, DAPI-Färbung, DAPI Bandpassfilter (Λem = 640 nm), 40x. Quantitative real-time RT-PCR In Abbildung 2 kann eine Down-Regulation der MDR1 mRNA von ca. 70% (schwarzer Balken) nach der Behandlung mit dem therapeutischen Bakterien gesehen werden. Parental-Zellen dienen als positive Kontrolle durch das Fehlen von MDR1-Überexpression. Die Zelllinie 257RDB P170 mit einem Plasmid, das anti-MDR1 shRNAs s als direkter Vergleich der transkingdom RNAi-Technologie mit anderen R genommenNAi silencing Strategien. Die unbehandelten resistent Zelllinie EPG85-257RDB überexprimierenden MDR1, die gleiche Zelllinie mit Bakterien ohne die shRNA exprimierenden Plasmid behandelt, und diese Zelllinie behandelt mit therapeutischen Bakterien, die ein Plasmid Ausdruck anti-MRP2 shRNAs wurden als positive Kontrollen. <img src="/files/ftp_upload/2099/2099fig2.jpg" alt="Abbildung 2" /> Abbildung 2: Quantitative real-time RT-PCR MDR1 mRNA-Expression nach Behandlung mit therapeutischen E. coli ceq221 Ausdruck anti-MDR1 shRNAs (MOI 1: 500). Die Normalisierung erfolgte mit dem Housekeeping-Gen Aldolase. Die MDR1/aldolase Verhältnis von unbehandelten Zellen der Zelllinie EPG85-257RDB wurden 100% gesetzt. P-Werte wurden mit dem Student-t-Test (* = p &lt;0,05, ** = p &lt;0,005, *** = p &lt;0,001). Western-Blot-Analyse Abbildung 3 zeigt, dass die MDR1 Downregulation erfolgte auch auf Protein-Ebene nach der bakteriellen Behandlung. Es zeigt die fehlende MDR1 Expression des Medikaments sensible Eltern Zelllinie (EPG85-257P), die MDR1 Expression der unbehandelten resistente Zelllinie (EPG85-257RDB) und der MDR1 Expression der behandelten Probe. Eine klare Down-Regulation der MDR1 der bakteriell behandelten Zellen beobachtet werden kann. <img src="/files/ftp_upload/2099/2099fig3.jpg" alt="Abbildung 3" /> Abbildung 3: Western-Blot-Analyse MDR1 Expression der unbehandelten Droge-sensitive EPG85-257P, das unbehandelte resistenten EPG-257RDB und der EPG85-257RDB nach Ko-Inkubation mit E.. coli ceq221 + p43 MDR1. Primäre Ab C219 1:100 und anti-Aktin 1:5 000, sekundäre Ab-Anti-Maus 1:10 000. Zytotoxizitätsassay Funktionelle Analyse wie die Zytotoxizitätsassay in Abbildung 4 dargestellt sind auch Indikatoren für das Funktionieren des transkingdom RNAi. Die elterliche Zelllinie und der Zell-Linie, die eine anit-MDR1 shRNA exprimierenden Plasmid zeigen keine Resistenz gegenüber Daunorubicin. Die resistenten Zelllinie EPG85-257RDB und zwei weitere Kontrollen keine signifikanten Änderungen des Widerstandes im Vergleich zu der Probe mit anti-MDR1 shRNA exprimierenden Bakterien, wo der Widerstand gegen Daunorubicin um ca. 90% rückgängig gemacht werden könnte behandelt. <img src="/files/ftp_upload/2099/2099fig4.jpg" alt="Abbildung 4" /> Abbildung 4: Zytotoxizitätsassay. Drug-spezifische IC50-Werte von einem Zytotoxizitätstest für das Überleben der Zelle bestimmt. P-Werte wurden mit dem Student-t-Test (* = p &lt;0,05, ** = p &lt;0,005, *** = p &lt;0,001). Anthrazyklin Anhäufung Test Nach Angaben der Abwehrschwäche von bakteriell behandelten Proben (Abbildung 4) wird die Anthrazyklin Ansammlung von anti-MDR1 shRNA behandelten Zellen um etwa 90% erhöht, wie in Abbildung 5 dargestellt. Die elterliche Zelllinie und die Zelllinie 257RDB P170 zeigen eine starke Daunorubicin Anhäufung von bis zu 100%. Die resistenten Variante zeigt nur selten eine Akkumulation. Zellen mit anti-MDR1 shRNA exprimierenden Bakterien behandelt zeigen eine Anthracyclin Anhäufung Steigerung von 90%. <img src="/files/ftp_upload/2099/2099fig5.jpg" alt="Abbildung 5" /> Abbildung 5: Anthrazyklin Anhäufung Assay. Anthrazyklin Ansammlung von Karzinomzellen 6 Tage nach der bakteriellen Behandlung mittels Durchflusszytometrie gemessen. P-Werte wurden mit dem Student-t-Test (* = p &lt;0,05, ** = p &lt;0,005, *** = p &lt;0,001).

<ol>
	<li>Kr&#252;hn, A., Wang, A., Fruehauf, J. H., Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+short+hairpin+RNAs+by+transkingdom+RNA+interference+modulates+the+classical+ABCB1-mediated+multidrug-resistant+phenotype+of+cancer+cells.">Delivery of short hairpin RNAs by transkingdom RNA interference modulates the classical ABCB1-mediated multidrug-resistant phenotype of cancer cells.</a> Cell Cycle. 8, 3349-3354 (2009).</li><li>Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MDR1/P-glycoprotein+(ABCB1)+as+target+for+RNA+interference-mediated+reversal+of+multidrug+resistance.">MDR1/P-glycoprotein (ABCB1) as target for RNA interference-mediated reversal of multidrug resistance.</a> Curr. Drug Targets. 7, 813-821 (2006).</li><li>Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+cancer+multidrug+resistance:+a+still+unsolved+problem.">An overview of cancer multidrug resistance: a still unsolved problem.</a> Cell. Mol. Life Sci. 65, 3145-3167 (2008).</li><li>Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+potential+of+RNA+interference+in+drug-resistant+cancers.">Therapeutic potential of RNA interference in drug-resistant cancers.</a> Fut. Oncol. 5, 169-185 (2009).</li><li>Nguyen, T. A., Fruehauf, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transkingdom+RNA+interference+(tkRNAi):+a+novel+method+to+induce+therapeutic+gene+silencing.">Transkingdom RNA interference (tkRNAi): a novel method to induce therapeutic gene silencing.</a> Methods Mol. Biol. 514, 27-34 (2009).</li><li>Nieth, C., Priebsch, A., Stege, A., Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+the+classical+multidrug+resistance+(MDR)+phenotype+by+RNA+interference+(RNAi.">Modulation of the classical multidrug resistance (MDR) phenotype by RNA interference (RNAi.</a> FEBS Lett. 545, 144-150 (2003).</li><li>Stege, A., Priebsch, A., Nieth, C., Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+and+complete+overcoming+of+MDR1/P-glycoprotein-mediated+multidrug+resistance+in+human+gastric+carcinoma+cells+by+RNA+interference.">Stable and complete overcoming of MDR1/P-glycoprotein-mediated multidrug resistance in human gastric carcinoma cells by RNA interference.</a> Cancer Gene Ther. 11, 699-706 (2004).</li><li>Stein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+in+vivo+reversal+of+the+multidrug+resistance+(MDR)+phenotype+by+jet-injection+of+anti-MDR1+short+hairpin+RNA-encoding+plasmid+DNA.">Complete in vivo reversal of the multidrug resistance (MDR) phenotype by jet-injection of anti-MDR1 short hairpin RNA-encoding plasmid DNA.</a> Mol. Ther. 16, 178-186 (2008).</li><li>Xiang, S., Fruehauf, J., Li, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+hairpin+RNA-expressing+bacteria+elicit+RNA+interference+in+mammals.">Short hairpin RNA-expressing bacteria elicit RNA interference in mammals.</a> Nat. Biotechnol. 24, 697-702 (2006).</li></ol>

Die Zellzahl für eine Infektion ausgesät und die entsprechenden MOI verwendet, sich kritisch mit den Zelllinien unter Beobachtung und deren Geschwindigkeit des Wachstums ab. Um eine optimale Zellzahl für die Aussaat sind pre-Experimente, um die Geschwindigkeit des Wachstums bestimmen dringend empfohlen. Daneben sollten verschiedene MOIs aufgrund der begrenzten getestet werden zu verlängern, um die Zellen können die bakterielle Invasion, ohne zu sterben von Stress stehen. Der optimale Zeitpunkt, wo die Down-Regulati...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Agar</td>
<td>&nbsp;</td>
<td>Biochrom AG</td>
<td>214050</td>
<td>&nbsp;</td>
<tr>
<td>Amphotericin B</td>
<td>&nbsp;</td>
<td>Biochrom AG</td>
<td>A2612</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco's Phosphate Buffered Saline-PBS (10 x)</td>
<td>&nbsp;</td>
<td>Invitrogen GmbH</td>
<td>14080048</td>
<td>&nbsp;</td>
<tr>
<td>E. coli ceq221</td>
<td>&nbsp;</td>
<td>Cequent Pharmaceuticals Inc.</td>
<td>&nbsp;</td>
<td>No catalogue available, direct order</td>
</tr>
<tr>
<td>Gentamycin</td>
<td>&nbsp;</td>
<td>Biochrom AG</td>
<td>A2710</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen GmbH</td>
<td>&nbsp;</td>
<td>Contains 5,000 units of penicillin (base) and 5,000 &mu;g of streptomycin (base)/ml utilizing penicillin G (sodium salt) and streptomycin sulfate in 0.85% saline.</td>
</tr>
<tr>
<td>Sodium chloride</td>
<td>&nbsp;</td>
<td>Merck KgaA</td>
<td>1064060500</td>
<td>&nbsp;</td>
<tr>
<td>Tryptone</td>
<td>&nbsp;</td>
<td>Difco Laboratories</td>
<td>211705</td>
<td>&nbsp;</td>
<tr>
<td>Yeast Extract</td>
<td>&nbsp;</td>
<td>Difco Laboratories</td>
<td>212750</td>
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bacterial delivery of rnai effectors: transkingdom rnai

RNA-Interferenz (RNAi) ist ein hoch wirksamer Mechanismus für die spezifische Hemmung der mRNA-Expression. Neben ihr Potenzial als ein leistungsfähiges Labor-Tool, erscheint der RNAi für therapeutische Nutzung vielversprechend. Für die Entwicklung der RNA-Interferenz (RNAi)-basierte Therapien, die Lieferung von RNAi-vermittelnde Agenten an Zielzellen ist eine der größten Hindernisse. Eine neuartige Strategie, diese Hürde zu überwinden, ist transkingdom RNAi (tk RNAi). Diese Technologie nutzt nicht-pathogene Bakterien, z. B. Escherichia coli, zu produzieren und zu liefern therapeutische kurze Haarnadel-RNA (shRNA) in Zielzellen zu RNAi zu induzieren. Eine der ersten Generation tk RNAi-vermittelnde Vektor, TRIP, enthält die Bakteriophagen T7 Promotor für die Expression Regulation eines therapeutischen shRNA von Interesse. Darüber hinaus hat die TRIP Inv locus von Yersinia pseudotuberculosis, dass Invasin kodiert, die natürliche invasive Bakterien erlaubt, β1-Integrin-positiven Säugetierzellen und die HlyA Gen aus Listeria monocytogenes, die Listeriolysin O. Dieses Enzym produziert eingeben kann die therapeutische shRNA zu entkommen Eintrag Vesikel im Zytoplasma der Zielzelle. TRIP-Konstrukte sind zu einem kompetenten nicht-pathogene Escherichia coli-Stamm, der T7-RNA-Polymerase, die für die T7-Promotor-gesteuerte Synthese von shRNAs kodiert eingeführt. Ein gut charakterisierter Krebs-assoziierte Zielmolekül für unterschiedliche RNAi-Strategien ist ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). Das ABC-Transporter fungiert als eine Droge Extrusionspumpe und vermittelt die &quot;klassische&quot; ABCB1-vermittelte Multidrug-Resistenz (MDR) Phänotyp von menschlichen Krebszellen, die durch eine spezifische Kreuzresistenz Muster geprägt ist. Verschiedene ABCB1-exprimierende MDR Krebszellen wurden mit anti-ABCB1 shRNA Expressionsvektor trägt E. behandelt coli. Dieses Verfahren führte zu einer Aktivierung der RNAi Wege innerhalb der Krebszellen und eine beträchtliche Down-Regulation der ABCB1 codierenden mRNA sowie den entsprechenden Medikamenten Extrusionspumpe. Dementsprechend wurde Kumulation in der unberührten resistente Krebszellen verbessert und die MDR Phänotyp wurde rückgängig gemacht. Durch dieses Modell die Daten liefern die Proof-of-concept, dass tk RNAi geeignet für die Modulation von Krebs-assoziierte Faktoren, z. B. ABCB1, in menschlichen Krebszellen ist.

Watch this Scientific Journal Video about Bacterial Delivery of RNAi Effectors: Transkingdom RNAi at JoVE.com

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

Für die Entwicklung der RNA-Interferenz (RNAi)-basierte Therapien, wurde eine neue Strategie entwickelt, transkingdom RNAi (tkRNAi). Diese Technologie nutzt nicht-pathogenen Bakterien zu produzieren und zu liefern therapeutische kurze Haarnadel-RNA (shRNA) in Zielzellen. Hier wurde tkRNAi erfolgreich Umkehrung der klassischen ABCB1-vermittelte Multidrug-Resistenz (MDR) von Krebszellen eingesetzt.

Bakterielle Lieferung von RNAi Effektoren: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory ...

bacterial-delivery-of-rnai-effectors-transkingdom-rnai

Research

JoVE Journal

Biology

13.4K Aufrufe.  Charité Campus Mitte. Für die Entwicklung der RNA-Interferenz (RNAi)-basierte Therapien, wurde eine neue Strategie entwickelt, transkingdom RNAi (tkRNAi). Diese Technologie nutzt nicht-pathogenen Bakterien zu produzieren und zu liefern therapeutische kurze Haarnadel-RNA (shRNA) in Zielzellen. Hier wurde tkRNAi erfolgreich Umkehrung der klassischen ABCB1-vermittelte Multidrug-Resistenz (MDR) von Krebszellen eingesetzt.

Serie: Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

Protocol for RNAi Assays in Adult Mosquitoes (A. gambiae)

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite.  The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Protocol for RNAi Assays in Adult Mosquitoes A. gambiae

Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite. The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Protokoll für die RNAi-Assays in Adult Moskitos (A. gambiae)

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video ...

Forschung

Biologie

Localized RNAi and Ectopic Gene Expression in the Medicinal Leech

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to perturb gene expression patterns in selected segments of leech embryos, leaving the untreated segments as internal controls.
The pneumatic capillary gun can be used to reach internal layers of cells at early stages of development without opening the specimen. As a method for localized introduction of substances into living tissues, the biolistic delivery with the gun has several advantages: it is fast, contact-free and non-destructive. In addition, a single capillary gun can be used for independent delivery of different substances. The delivery region can have lateral dimensions of ~50-150 &#181;m and extends over ~15 &#181;m around the mean penetration depth, which is adjustable between 0 and 50 &#181;m. This delivery has the advantage of being able to target a limited number of cells in a selected location intermediate between single cell knock down by microinjection and systemic knockdown through extracellular injections or by means of genetic approaches.
For knocking down or knocking in the expression of the axon guidance molecule Netrin, which is naturally expressed by some central neurons and in the ventral body wall, but not the dorsal domain, we deliver molecules of dsRNA or plasmid-DNA into the body wall and central ganglia. This procedure includes the following steps: (i) preparation of the experimental setup for a specific assay (adjusting the accelerating pressure), (ii) coating the particles with molecules of dsRNA or DNA, (iii) loading the coated particles into the gun, up to two reagents in one assay, (iv) preparing the animals for the particle delivery, (v) delivery of coated particles into the target tissue (body wall or ganglia), and (vi) processing the embryos (immunostaining, immunohistochemistry and neuronal labeling) to visualize the results, usually 2 to 3 days after the delivery.
When the particles were coated with netrin dsRNA, they caused clearly visible knock-down of netrin expression that only occurred in cells containing particles (usually, 1-2 particles per cell). Particles coated with a plasmid encoding EGFP induced fluorescence in neuronal cells when they stopped in their nuclei.

In this video, we show a procedure for an accurate biolistic delivery of reagents into live tissue with a novel miniature gene gun. We are knocking down the expression of the axon guidance molecule Netrin in leech embryos by delivering molecules of dsRNA into the ventral body wall and ganglia of single segments.

Lokalisierte RNAi und ektopische Genexpression in der Blutegel

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to ...

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

Identification of Growth Inhibition Phänotypen durch Expression von bakteriellen Typ III Effektoren in Hefe Induced

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

MISSION esiRNA für RNAi-Screening in Säugerzellen

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

RNAi Interference by dsRNA Injection into Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.
Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.

RNAi-Interferenz durch dsRNA Injektion in Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans3, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis4 and Pristionchus pacificus5. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior6-7, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers5. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis4 and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms8. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F1 progeny. Unlike C. elegans, but like most other nematodes, P. pacificus PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4 promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92) mutant in a visually informative protocol.

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

In model organisms, transgenesis can manipulate gene functions while RNAi can knockdown specific mRNA transcripts 1-2. This protocol aims to illustrate the techniques needed to introduce stably transmitted DNA and transient double stranded RNA into the necromenic nematode Pristionchus pacificus for studies in evolutionary, developmental, and behavioral biology.

RNAi-vermittelte Gene Knockdown und Transgenese durch Mikroinjektion in die Necromenic Nematode Pristionchus pacificus</em

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi-Screening auf postembryonalen Phänotypen in Identifizieren C elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

Quantitative und automatisierte Hochdurchsatz-genomweiten RNAi-Screens in C elegans</em

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. 
This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis1. 
The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

In this protocol we combine RNAi-mediated gene silencing with an in vivo diuresis assay to study the effects knockdown of genes of interest has on mosquito fluid excretion.

RNAi-vermittelte Gen-Knockdown und<em&gt; In Vivo</em&gt; Diurese Assay in Adult Female<em&gt; Aedes aegypti</em&gt; Moskitos

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion ...

Phosphopeptide Analysis of Rodent Epididymal Spermatozoa

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once the pre-cursor round cell begins to elongate and differentiate into what is morphologically recognized as a spermatozoon. However, the spermatozoon is very immature, having no ability for motility or egg recognition. Both of these events occur once the spermatozoa transit a secondary organ known as the epididymis. During the ~12 day passage that it takes for a sperm cell to pass through the epididymis, post-translational modifications of existing proteins play a pivotal role in the maturation of the cell. One major facet of such is protein phosphorylation. In order to characterize phosphorylation events taking place during sperm maturation, both pure sperm cell populations and pre-fractionation of phosphopeptides must be established. Using back flushing techniques, a method for the isolation of pure spermatozoa of high quality and yield from the distal or caudal epididymides is outlined. The steps for solubilization, digestion, and pre-fractionation of sperm phosphopeptides through TiO2 affinity chromatography are explained. Once isolated, phosphopeptides can be injected into MS to identify both protein phosphorylation events on specific amino acid residues and quantify the levels of phosphorylation taking place during the sperm maturation processes.

Proteomic analysis of any cell type is highly dependent on both purity and pre-fractionation of the starting material in order to de-complexify the sample prior to liquid chromatography mass spectrometry (MS). By using back-flushing techniques, pure spermatozoa can be obtained from rodents. Following digestion, phosphopeptides can be enriched using TiO2.

Phosphopeptid Analyse der Nebenhoden Nagetier Spermien

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once ...