El trabajo fue financiado por el subsidio no. 01GU0615 del &quot;Bundesministerium für Forschung und Technologie de Alemania (BMBF).

1) Entrega bacteriana de shRNAs <ol><li> Antes de comenzar con el cultivo bacteriano, uno tiene que preparar LB-medio y LB-agar. </li><li> Para el LB-medio pesar de extracto de levadura (0,5% w / v), Bacto triptona (1,0% w / v) y NaCl (0,6% w / v) y diluir estos componentes en agua bidestilada. Las soluciones preparadas deben ser esterilizados en autoclave y luego se vaya a utilizar. </li><li> Para las placas de agar-agar LB bacto (1,5% w / v) se debe añadir a la LB-medio anteriores a la esterilización. </li><li> Calentar el LB-agar en el microondas hasta que el LB-agar se disuelva por completo. Deje que la solución se enfríe hasta que la botella se puede tocar con facilidad sin quemarse. Añadir a la kanamicina (100 mg / ml) para la selección de clones positivos en los siguientes pasos. </li><li> Preparar agar LB-placas en un banco de flujo laminar (condiciones estériles) con la pipeta 20 ml de la solución de LB-agar en 10 cm de placas Petri. Deje que las placas de enfriamiento hasta que el líquido se ha convertido en sólidos. Las placas están listas para usar y se puede almacenar a 4 ° C. </li><li> ARNhc de codificación de vectores de expresión se transformó en E. competentes ceq221 coli por choque térmico mediante el procedimiento de CaCl2. </li><li> Placa de las bacterias transformadas en placas de agar LB que contienen 100 mg / ml de kanamicina, cerrar la tapa, sellar las placas con parafilm y cultivar al revés a 37 ° C durante la noche. </li><li> Inocular bacterias culturas mini clones positivos recogiendo colonias crecido con un palillo. Transferir el diente recoger en 7,0 ml de agua dulce LB-medio que contiene 100 mg / ml de kanamicina y cultivar a 37 ° C durante la noche en un agitador a 200 rpm. </li><li> Inocular 100 ml de agua dulce-LB medio que contiene 100 mg / ml kanamicina 1:100 con la cultura de la noche a través de mini en un 1 l erlenmeyer, y se incuba a 37 ° C durante la noche en un agitador a 200 rpm. </li><li> Semillas de 25 x 10 4 (número de células sembradas depende de la velocidad de crecimiento de las células de la línea celular de acuerdo; confluencia debe ser ~ 70-80% 24 horas después de la siembra) las células humanas del carcinoma gástrico (EPG85-257RDB) por pocillo de 6 - platos bien e incubar estas a 37 ° C, en una atmósfera de 5% CO 2, vapor de agua saturado durante la noche. </li><li> Anterior a la infección de las células cancerosas, sobre las culturas noche de los conocimientos tradicionales RNAi vector que contiene E. coli se miden en un fotómetro para determinar el OD 600. Diluir la solución bacteriana hasta una densidad óptica de 0,5 se llega a (OD 600 = 0,5 equivale a 1,6 x 10 8 células bacterianas / ml). </li><li> Vuelva a colocar el medio celular que contiene FCS-cultura de las células de carcinoma contra FCS medio libre de 30 minutos anteriores a las bacterias co-incubación. </li><li> Lávese las bacterias dos veces con 1 x PBS, y se diluye en suero libre de Leibovitz medio L-15. </li><li> Añadir bacterias diluido a las células cancerosas en que desee MOI (multiplicidad de infección = número de células bacterianas por células cancerosas) y las bacterias co-incubación y las células cancerosas durante 2 horas a 37 º C. </li><li> Después de 2 horas de las células del cáncer co-incubación se lavaron dos veces con 1 x PBS y una vez con el suero que contienen Leibovitz L-15 suplementado con 100 U / ml de penicilina, 100 mg / ml de estreptomicina, 2.5 mg / ml de anfotericina, 150 mg / ml de gentamicina, y 100 ug / ml de kanamicina. </li><li> Continuar con el cultivo de las células cancerosas a 37 ° C, en una atmósfera de 5% CO 2, vapor de agua saturada. </li><li> Los efectos del tratamiento bacteriano puede ser visualizado por microscopía de fluorescencia, medidos por PCR cuantitativa en tiempo real RT en el nivel de ARNm y por Western blot en el nivel de proteínas, y se muestra con ensayos funcionales como el análisis de FACS, y ensayos de proliferación celular. (Si se desea, estos procedimientos pueden ser descritos en detalle). </li></ol> 2) Los resultados representativos Si el protocolo se realiza correctamente, los resultados deben ser comparables a las que se muestran a continuación. Microscopía de fluorescencia La figura 1 muestra una célula humana carcinoma gástrico tres horas después del tratamiento bacteriano (a) en comparación con una célula no tratada (b). Alrededor del núcleo de la célula tratada, las bacterias pueden ser detectados. <img src="/files/ftp_upload/2099/2099fig1.jpg" alt="Figura 1" /> Figura 1: microscopía de fluorescencia de células humanas de carcinoma gástrico después del tratamiento bacteriano (1:500) y un sin tratar de células humanas de carcinoma gástrico como el control, DAPI mancha, DAPI banda pasante del filtro (Λem = 640 nm), el objetivo de 40x. Cuantitativa en tiempo real RT-PCR En la figura 2 una regulación a la baja de MDR1 ARNm de aproximadamente 70% (rayo negro) se puede ver después del tratamiento con las bacterias terapéuticas. Células de los padres sirven como control positivo debido a la falta de MDR1 sobre-expresión. La línea celular 257RDB p170 que contiene un plásmido que expresa anti-MDR1 shRNAs s tomado como una comparación directa de la tecnología de RNAi transkingdom a otras organizaciones de INAi silenciar las estrategias. La línea de células resistentes no tratados EPG85-257RDB sobreexpresión de MDR1, la misma línea celular tratados con bacterias que carecen de la expresión del shRNA plásmido, y esta línea de células tratadas con bacterias terapéuticas que lleva un plásmido que expresa anti-MRP2 shRNAs fueron tomados como controles positivos. <img src="/files/ftp_upload/2099/2099fig2.jpg" alt="Figura 2" /> Figura 2: cuantitativa en tiempo real RT-PCR la expresión del ARNm MDR1 después del tratamiento con fines terapéuticos E. coli ceq221 expresar anti-MDR1 shRNAs (MOI 1: 500). La normalización se realizó con la aldolasa limpieza de genes. La relación MDR1/aldolase de las células no tratadas de la línea celular EPG85-257RDB se crearon 100%. P-valores se calcularon utilizando la t de Student-test (* = p &lt;0,05, ** = p &lt;0,005, *** = p &lt;0,001). Western blot La figura 3 muestra que el MDR1 por la regulación también se llevó a cabo en el nivel de proteína bacteriana después del tratamiento. Esto demuestra la falta de MDR1 expresión de la droga sensible a la línea celular parental (EPG85-257P), la expresión de MDR1 de la línea celular de drogas sin tratamiento resistente (EPG85-257RDB) y la expresión de MDR1 de la muestra tratada. Una regulación clara abajo de MDR1 de las células tratadas con bacterias se pueden observar. <img src="/files/ftp_upload/2099/2099fig3.jpg" alt="Figura 3" /> Figura 3: Análisis de Western blot MDR1 los niveles de expresión de los no tratados con drogas sensibles EPG85-257P, el grupo no tratado resistente a los medicamentos EPG-257RDB, y de EPG85-257RDB después de co-incubación con E.. coli ceq221 + P43 MDR1. Ab primaria C219 1:100 y anti-actina 1:5 000, Ab secundario anti-ratón de 1:10 000. Citotoxicidad ensayo El análisis funcional como el ensayo de citotoxicidad se muestra en la figura 4 también son indicadores para el funcionamiento de la RNAi transkingdom. La línea celular de sus padres y la línea celular que contiene un ARNhc anit-MDR1 expresar plásmido no muestran resistencia a la daunorubicina. La línea celular resistente EPG85-257RDB y dos controles posteriores no mostraron cambios significativos en la resistencia en comparación con la muestra tratada con anti-bacterias del shRNA expresar MDR1 donde podría ser la resistencia a la daunorubicina invertido en un 90%. <img src="/files/ftp_upload/2099/2099fig4.jpg" alt="Figura 4" /> Figura 4: ensayo de citotoxicidad. Drogas específicas IC50-valores determinados por un ensayo de citotoxicidad para la supervivencia celular. P-valores se calcularon utilizando la t de Student-test (* = p &lt;0,05, ** = p &lt;0,005, *** = p &lt;0,001). Antraciclina ensayo de acumulación De acuerdo con la disminución de la resistencia bacteriana de las muestras tratadas (Figura 4), la acumulación de antraciclina de anti-células MDR1 siRNA tratados se incrementa alrededor de un 90% como se muestra en la figura 5. La línea celular de sus padres y la línea celular 257RDB p170 muestran una acumulación daunorubicina fuerte hasta el 100%. La variante resistente a la muestra suele haber acumulación. Las células tratadas con anti-bacterias MDR1 ARNhc expresar muestran un aumento de la acumulación de antraciclina de 90%. <img src="/files/ftp_upload/2099/2099fig5.jpg" alt="Figura 5" /> Figura 5: ensayo de antraciclina de acumulación. Antraciclina acumulación de células de carcinoma de seis días después del tratamiento bacteriano mediante citometría de flujo. P-valores se calcularon utilizando la t de Student-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>

El número de células sembradas para la infección y se utiliza el MOI correspondientes, dependen fundamentalmente de las líneas celulares en observación y su velocidad de crecimiento. Para encontrar números de celular óptimo para la siembra, pre-experimentos para determinar la velocidad de crecimiento son muy recomendables. Además de esto, las MOI diferentes deben ser probados debido a la limitada se extiende a que las células pueden resistir la invasión bacteriana sin morir de estrés. El punto óptimo de tiem...

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		<div>
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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>
<td>&nbsp;</td>		</tbody>
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bacterial delivery of rnai effectors: transkingdom rnai

ARN de interferencia (ARNi) es un mecanismo altamente efectivo para la inhibición específica de la expresión del ARNm. Además de su potencial como herramienta de laboratorio poderosa, la vía de RNAi parece ser prometedora para la utilización terapéutica. Para el desarrollo de la interferencia de ARN (RNAi), las terapias basadas en la entrega de RNAi agentes mediadores de las células diana es uno de los principales obstáculos. Una nueva estrategia para superar este obstáculo es transkingdom RNAi (tk RNAi). Esta tecnología utiliza bacterias no patógenas, por ejemplo, Escherichia coli, para producir y entregar terapéutica de horquilla corta RNA (shRNA) en las células diana para inducir RNAi. Una primera generación de los conocimientos tradicionales RNAi mediar vector, viaje, contiene el promotor del bacteriófago T7 para la regulación de la expresión de un ARNhc terapéuticas de interés. Además, el lugar ha VIAJE Inv de Yersinia pseudotuberculosis que codifica invasina, lo que permite bacterias naturales no invasivas para entrar β1-integrina-positivo células de mamíferos y el gen HlyA de Listeria monocytogenes, que produce listeriolisina O. Esta enzima permite la shRNA terapéutica para escapar de la entrada de vesículas en el citoplasma de la célula diana. Construye TRIP se introducen en un tribunal competente no patógena cepa de Escherichia coli, que codifica la ARN polimerasa T7 necesarios para la T7 promotor impulsado por la síntesis de shRNAs. A bien caracterizados asociados con el cáncer molécula diana de las diferentes estrategias de RNAi es ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). Esta ABC-transportador actúa como una bomba de extrusión de drogas y media la &quot;clásica&quot; ABCB1 mediada por la resistencia a múltiples fármacos (MDR) fenotipo de las células de cáncer humano que se caracteriza por un patrón de resistencia cruzada específica. ABCB1 diferentes que expresan las células cancerosas MDR fueron tratados con anti-ABCB1 del shRNA vector de expresión E. rodamientos coli. Este procedimiento dio lugar a la activación de las vías de ARNi en las células cancerosas y una regulación considerable abajo del ARNm que codifica ABCB1, así como la bomba de extrusión de drogas correspondiente. En consecuencia, la acumulación del fármaco fue mayor en las células cancerosas resistentes a los medicamentos prístina y el fenotipo MDR fue revocada. Por medio de este modelo los datos que presente la prueba de concepto que los conocimientos tradicionales RNAi es adecuado para la modulación de los factores asociados con el cáncer, por ejemplo, ABCB1, en ​​células de cáncer humano.

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

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

Para el desarrollo de la interferencia de ARN (RNAi), las terapias basadas en una nueva estrategia se ha desarrollado, transkingdom RNAi (tkRNAi). Esta tecnología utiliza bacterias no patógenas para producir y entregar terapéutica de horquilla corta RNA (shRNA) en las células diana. Aquí, tkRNAi se aplicó con éxito para la reversión de la resistencia a múltiples fármacos clásicos ABCB1 mediada (MDR) de las células cancerosas.

Entrega bacteriana de RNAi efectores: 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 vistas.  Charité Campus Mitte. Para el desarrollo de la interferencia de ARN (RNAi), las terapias basadas en una nueva estrategia se ha desarrollado, transkingdom RNAi (tkRNAi). Esta tecnología utiliza bacterias no patógenas para producir y entregar terapéutica de horquilla corta RNA (shRNA) en las células diana. Aquí, tkRNAi se aplicó con éxito para la reversión de la resistencia a múltiples fármacos clásicos ABCB1 mediada (MDR) de las células cancerosas.

Video: 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.

Protocolo para ensayos de RNAi en mosquitos adultos (A. gambiae)

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

Investigación

Biología

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.

Localizada RNAi y la expresión génica ectópico en la sanguijuela medicinal

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.

Identificación de los fenotipos de inhibición del crecimiento inducida por la expresión de tipo bacteriana efectores III en la levadura

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.

MISIÓN esiRNA sobre el cribado del ARNi en células de mamífero

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 interferencia por inyección de dsRNA en Drosophila Los embriones

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 mediada por derribo de genes y transgénesis por microinyección en el nematodo Necromenic 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 de detección para identificar fenotipos postembrionarios en 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.

Cuantitativos y automatizada de alto rendimiento del genoma completo de RNAi pantallas en 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 mediada por Gene y Derribo<em&gt; En Vivo</em&gt; Ensayo de diuresis en Mujeres Adultas<em&gt; Aedes aegypti</em&gt; Los mosquitos

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.

Análisis Phosphopeptide de roedores epidídimo Espermatozoides

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