Trabalho foi financiado por bolsa nenhuma. 01GU0615 do &quot;Bundesministerium für Forschung und Technologie (BMBF).

1) Entrega bacteriana da shRNAs <ol><li> Antes de iniciar com a cultura bacteriana, um tem que se preparar LB médio e LB-ágar. </li><li> Para o LB médio pesar extrato de levedura (0,5% w / v), Bacto triptona (1,0% w / v) e NaCl (0,6% w / v) e diluir estes componentes em água bidest. As soluções preparadas têm que ser esterilizados em autoclave e são, então, pronto para uso. </li><li> Para LB-agar agar Bacto placas (1,5% w / v) tem de ser adicionado à LB médio anteriores à esterilização. </li><li> Aquecer LB-ágar no microondas até que o LB-ágar esteja completamente dissolvido. Deixe a solução esfriar até que a garrafa pode ser tocado facilmente sem se queimar. Adicionar canamicina (100 mg / ml) para a seleção de clones positivos em outras etapas. </li><li> Prepare LB-ágar placas em um banco de fluxo laminar (condições estéreis) pipetando 20 ml da solução de LB-ágar em 10 cm de Petri-pratos. Deixe arrefecer as placas até que o líquido tornou-se sólido. As placas estão agora prontos para usar e pode ser armazenado a 4 ° C. </li><li> Vetores shRNA codificação expressão são transformados em E. competente ceq221 coli por choque térmico usando o procedimento de CaCl 2. </li><li> A placa de bactérias transformadas em LB-ágar placas contendo 100 mg / ml de canamicina, feche a tampa, selar as placas com parafilme e cultivar de cabeça para baixo a 37 ° C durante a noite. </li><li> Inocular culturas bacterianas com mini-clones positivos escolhendo colônias crescidas com uma picareta do dente. Transferir o palito em 7,0 ml de LB fresco médio, contendo 100 mg / ml kanamicina e cultivar a 37 ° C durante a noite em um shaker a 200 rpm. </li><li> Inocular 100 ml de LB fresco médio, contendo 100 mg / ml kanamicina 1:100 com a cultura ao longo da noite em um mini-1 frasco Erlenmeyer l, e incubar a 37 ° C durante a noite em um shaker a 200 rpm. </li><li> Semente de 25 x 10 4 (número de células inoculadas depende da velocidade do crescimento das células da linha celular de acordo; confluência deve ser 70-80% ~ 24 horas após a semeadura) células de carcinoma gástrico (EPG85-257RDB) por poço em 6 - pratos bem e incubar estes em 37 ° C, em um CO 2, 5% de água atmosfera saturada de vapor durante a noite. </li><li> Anterior à infecção de células cancerosas, ao longo culturas noite de tk RNAi vector contendo E. coli são medidos em um fotômetro para determinar a OD 600. Diluir a solução de bactérias até uma densidade óptica de 0,5 é atingido (OD 600 = 0,5 é igual a 1,6 x 10 8 células bacterianas / ml). </li><li> Substituir o FCS contendo meio de cultura de células do carcinoma de células contra FCS meio livre 30 min anterior para bacterianas co-incubação. </li><li> Lavar as bactérias duas vezes com 1 x PBS, e diluir em soro livre de Leibovitz L-15 médio. </li><li> Adicionar bactérias diluído para as células cancerosas na desejado MOI (multiplicidade de infecção = número de células bacterianas por célula de câncer) e co-incubação de bactérias e células de câncer por 2 horas a 37 ° C. </li><li> Após 2 horas de células co-incubação de câncer foram lavadas duas vezes com PBS 1x x e uma vez com o soro contendo Leibovitz L-15 meio suplementado com 100 u / ml de penicilina, estreptomicina 100 mg / ml, 2,5 mg / ml anfotericina, 150 mg / ml de gentamicina, e 100 mg / ml de canamicina. </li><li> Continuar a cultivar as células cancerosas a 37 ° C, em um CO 2, 5% de água atmosfera de vapor saturado. </li><li> Os efeitos do tratamento de bactérias pode ser visualizada por microscopia fluorescente, medido pelo quantitativo em tempo real RT PCR em nível de mRNA, e pela análise de western blot em nível de proteína, e mostra com ensaios funcionais como FACS, análise e ensaios de proliferação celular. (Se queria, estes procedimentos podem ser descritos em detalhe). </li></ol> 2) Resultados Representante Se o protocolo for realizado corretamente, os resultados devem ser comparáveis ​​aos mostrado abaixo. Microscopia de fluorescência A Figura 1 mostra uma célula de carcinoma gástrico humano três horas após o tratamento bacteriana (a) em comparação com uma célula não tratada (b). Ao redor do núcleo da célula tratada, as bactérias podem ser detectadas. <img src="/files/ftp_upload/2099/2099fig1.jpg" alt="Figura 1" /> Figura 1: microscopia fluorescente de células de carcinoma gástrico Humanos após o tratamento de bactérias (1:500) e uma célula de carcinoma gástrico humano sem tratamento como controle, DAPI-coloração, DAPI bandpass filter (Λem = 640 nm), objetiva de 40x. Quantitativa em tempo real RT PCR Na figura 2 um regulamento para baixo de MDR1 mRNA de cerca de 70% (feixe de preto) pode ser visto após o tratamento com a bactéria terapêutico. Células dos pais servem como um controle positivo, devido à falta de MDR1 superexpressão. A linhagem celular 257RDB p170 contendo um plasmídeo expressando anti-MDR1 shRNAs s tomado como comparação direta da tecnologia de RNAi para R transkingdom outrosNAi silenciar estratégias. A linhagem de células resistentes tratadas EPG85-257RDB superexpressão MDR1, a linha de celular mesmo tratados com bactérias sem o plasmídeo shRNA expressar, e esta linhagem de células tratadas com bactérias terapêutica carregando um plasmídeo expressando anti-MRP2 shRNAs foram tomados como controles positivos. <img src="/files/ftp_upload/2099/2099fig2.jpg" alt="Figura 2" /> Figura 2: quantitativa em tempo real RT PCR a expressão do mRNA MDR1 após o tratamento com E. terapêutica ceq221 coli expressando anti-MDR1 shRNAs (MOI 1: 500). Normalização foi realizada através do aldolase gene housekeeping. A relação MDR1/aldolase de células não tratadas da linha celular EPG85-257RDB foram criados 100%. P-valores foram calculados usando o teste t de Student (* = p &lt;0,05, ** = p &lt;0,005, *** = p &lt;0,001). Ocidental blot Figura 3 revela que a MDR1 regulação também ocorreu no nível de proteína após o tratamento de bactérias. Ela mostra a falta de expressão da MDR1 linhagem de células de drogas sensíveis parental (EPG85-257p), a expressão da MDR1 não tratada linhagem de células resistentes aos medicamentos (EPG85-257RDB) ea expressão MDR1 da amostra tratada. A regulamentação clara para baixo de MDR1 das células tratadas com bactérias pode ser observada. <img src="/files/ftp_upload/2099/2099fig3.jpg" alt="Figura 3" /> Figura 3: análise de Western blot MDR1 níveis de expressão do tratado de drogas sensíveis EPG85-257p, a não tratada resistentes a drogas EPG-257RDB, e de EPG85-257RDB após co-incubação com E.. coli ceq221 + p43 MDR1. Principal Ab C219 1:100, e anti-actina 1:5 000, secundário Ab anti-mouse 1:10 000. Ensaio de citotoxicidade Análise funcional como o ensaio de citotoxicidade mostrado na figura 4 também são indicadores para o funcionamento do RNAi transkingdom. A linhagem de células parentais e da linha de célula que contém um shRNA anit-MDR1 expressar plasmídeo não mostram qualquer resistência à daunorrubicina. A linhagem de células resistentes EPG85-257RDB e dois controles ainda não mostram mudanças significativas na resistência em comparação com a amostra tratada com anti-bactérias MDR1 shRNA expressando onde a resistência à daunorrubicina pode ser revertido em cerca de 90%. <img src="/files/ftp_upload/2099/2099fig4.jpg" alt="Figura 4" /> Figura 4: Ensaio de Citotoxicidade. Drogas específicas IC50-valores determinados por um ensaio de citotoxicidade para a sobrevivência da célula. P-valores foram calculados usando o teste t de Student (* = p &lt;0,05, ** = p &lt;0,005, *** = p &lt;0,001). Antraciclina ensaio de acumulação De acordo com a baixa resistência de amostras de bactérias tratada (figura 4), o acúmulo de células antraciclina anti-MDR1 shRNA tratada é aumentada em cerca de 90%, como mostrado na figura 5. A linhagem de células parentais e as linha celular 257RDB p170 mostram uma forte acumulação daunorrubicina até 100%. A variante resistente mostra raramente qualquer acumulação. Células tratadas com anti-bactérias MDR1 shRNA expressar mostram um aumento de acumulação anthracyline de 90%. <img src="/files/ftp_upload/2099/2099fig5.jpg" alt="Figura 5" /> Figura 5: ensaio de antraciclina acumulação. Antraciclina acúmulo de células de carcinoma seis dias após o tratamento de bactérias medida por citometria de fluxo. P-valores foram calculados usando o teste t de Student (* = 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>

Não há conflitos de interesse declarados.

O número de células semeadas para a infecção e os MOI correspondentes utilizados, dependem criticamente as linhas de células sob observação e sua velocidade de crescimento. Para encontrar o número de células ideal para a semeadura, a pré-experimentos para determinar a velocidade de crescimento são fortemente recomendados. Além disso, MOIS diferentes devem ser testados, devido à limitada estender-se que as células podem suportar a invasão bacteriana sem morrer de stress. O ponto ótimo de tempo em que a re...

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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

Interferência de RNA (RNAi) representa um mecanismo de alto eficaz para inibição específica de expressão de mRNA. Além de seu potencial como uma ferramenta poderosa de laboratório, a via RNAi parece ser promissor para uso terapêutico. Para o desenvolvimento de interferência de RNA (RNAi) baseado em terapias, a entrega de RNAi-agentes mediadores de células-alvo é um dos grandes obstáculos. A nova estratégia para superar este obstáculo é transkingdom RNAi (tk RNAi). Esta tecnologia utiliza bactérias não-patogênicas, por exemplo, Escherichia coli, para produzir e entregar terapêutica curto hairpin RNA (shRNA) em células-alvo para induzir RNAi. A primeira geração tk RNAi-mediadora vector TRIP, contém o bacteriófago T7 promoter de regulação da expressão de um shRNA terapêutico de interesse. Além disso, TRIP tem o locus de Inv Yersinia pseudotuberculosis, que codifica invasin, que permite que as bactérias naturais não-invasivo para entrar β1 positiva de integrina células de mamíferos eo gene HlyA de Listeria monocytogenes, que produz listeriolisina O. Esta enzima permite que o shRNA terapêuticas para escapar entrada vesículas no citoplasma da célula-alvo. Constrói TRIP são introduzidos em um competente coli cepa não-patogênica coli, que codifica T7 RNA polimerase necessário para a síntese T7 promoter-driven de shRNAs. A molécula-alvo bem caracterizado de câncer associados para diferentes estratégias RNAi é ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). Esta ABC-transportador atua como uma bomba de extrusão de drogas e media o &quot;clássico&quot; resistência a múltiplas drogas ABCB1 mediada (MDR) fenótipo de células de câncer humano, que é caracterizado por um padrão específico de resistência cruzada. ABCB1 expressando diferentes células de câncer de MDR foram tratados com anti-ABCB1 expressão shRNA vector rolamento E. coli. Este procedimento resultou na ativação das vias RNAi nas células do câncer e de um regulamento considerável para baixo do mRNA codificação ABCB1, bem como a bomba de extrusão correspondente drogas. Assim, a acumulação de drogas foi reforçada no pristine células resistentes a drogas câncer eo fenótipo MDR foi revertida. Por meio deste modelo os dados fornecem a prova de conceito que tk RNAi é adequado para a modulação de câncer de fatores associados, como por exemplo ABCB1, em células de câncer 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 o desenvolvimento de interferência de RNA (RNAi) baseado em terapias, uma nova estratégia foi desenvolvida, transkingdom RNAi (tkRNAi). Esta tecnologia utiliza bactérias não-patogênicas de produzir e entregar terapêutica curto hairpin RNA (shRNA) em células-alvo. Aqui, tkRNAi foi aplicado com sucesso para a reversão de multirresistência clássica ABCB1 mediada (MDR) de células cancerosas.

Entrega bacteriana de RNAi Effectors: 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 visualizações.  Charité Campus Mitte. Para o desenvolvimento de interferência de RNA (RNAi) baseado em terapias, uma nova estratégia foi desenvolvida, transkingdom RNAi (tkRNAi). Esta tecnologia utiliza bactérias não-patogênicas de produzir e entregar terapêutica curto hairpin RNA (shRNA) em células-alvo. Aqui, tkRNAi foi aplicado com sucesso para a reversão de multirresistência clássica ABCB1 mediada (MDR) de células cancerosas.

Vídeo: 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 Ensaios RNAi em mosquitos adultos (A. gambiae)

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

Biologia

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 e Expressão Gênica ectópica na sanguessuga 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.

Identificação de fenótipos inibição do crescimento induzido pela expressão de Effectors Tipo III bacteriana em 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 ...

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.

EsiRNA MISSÃO para a seleção RNAi em células de mamíferos

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 Interferência por injeção dsRNA em Drosophila Os embriões

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 Mediated Gene Knockdown e Transgénese por microinjeção no Nematóides 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 triagem para identificar fenótipos pós-embrionário em 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.

Quantitativos e automatizada de alto rendimento de todo o genoma Telas de RNAi em 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 knockdown do gene e<em&gt; In Vivo</em&gt; Ensaio diurese no Feminino Adulto<em&gt; Aedes aegypti</em&gt; Os 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.

Fosfopéptido Análise de Rodent epidídimo Espermatozóides

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