Ringraziamo i membri del nostro laboratorio per le discussioni fruttuose e assistenza tecnica. Questa presentazione video è stato sostenuto dal Preside Associato per la Ricerca e il Dipartimento di Veterinaria Patobiologia, Centro per la Veterinaria Scienze della Salute, Oklahoma State University. La ricerca è stata finanziata dal Ministerio de Ciencia e Innovación, Spagna (progetto BFU2008-01244/BMC), il progetto CSIC intramurale PA1002451 di JF, Walter R. Sitlington Dotato Cattedra per la ricerca alimentare animale a KMK, CVHS 2009 RAC concessione, OAE Fondi sanità animale e USDA, Nazionale di Grant Research Initiative competitivi, n. 2007-04613.

1. Generazione di dsRNA. <ol><li> Sintetizzare primer oligonucleotidi contenenti sequenze promotore T7 per la trascrizione in vitro e la sintesi di dsRNA (ad esempio, per Dermacentor variabilis subolesin utilizzare primer oligonucleotidi D8AAT75: 5&#39;-TAATACGACTCACTATAGGGTACTGACTGGGATCCCCTGCACAGT-3 &#39;e D8DVT73: 5&#39;-TAATACGACTCACTATAGGGTACTCGAGCTTGGTGGAAAGGACG-3&#39;). </li><li> Amplificare gene bersaglio mediante RT-PCR utilizzando 10 pmol di ciascun primer oligonucleotidi e 1-10 ng di RNA totale tick. </li><li> Purificare il prodotto della PCR. </li><li> Sintetizzare dsRNA utilizzando 8 ml di prodotto purificato PCR. </li><li> Quantificare dsRNA mediante spettrometria. </li></ol> 2. Iniezione di zecche con dsRNA. 2.1. Preparazione di zecche per l&#39;iniezione. <ol><li> In primo luogo, lavare le zecche in una serie di soluzioni agitando in ogni soluzione in una provetta da 50 ml per centrifuga monouso, decantazione la soluzione attraverso uno schermo sottile rete metallica sopra la parte superiore del tubo per mantenere le zecche. La sequenza delle soluzioni per le zecche lavaggio è l&#39;acqua del rubinetto, il perossido di idrogeno al 3%, due lavaggi di acqua distillata, 70% di etanolo e altri due lavaggi con acqua distillata. </li><li> Blot-asciugare le zecche su carta assorbente. </li><li> Contare le zecche in gruppi da 20 a 50, a seconda della sperimentazione, posto le zecche di ogni gruppo in una tazza di 1,25 once di plastica con un coperchio a tenuta, arredata e un&#39;etichetta con il numero del gruppo sperimentale. </li></ol> 2.2. Tick ​​squadra iniezione. Il team di RNAi è composto da tre persone: (1) una persona che ha posizioni di ogni segno di spunta sul doppio nastro adesivo apposto su un foglio di rosso per impronte dentarie, (2) una persona che inietta le zecche e (3) una persona che controlla le zecche dopo iniezione, respira di CO 2 sul zecche per attivarli e conta le zecche che vivono nelle tazze etichettata con il numero del gruppo sperimentale. Tutti i membri della squadra devono indossare guanti monouso. 2.3. Il posizionamento di zecche per l&#39;iniezione. <ol><li> Cattura un segno di spunta utilizzando pinze Dumont bene e posizionarlo sul lato ventrale doppio nastro adesivo apposto su un foglio 3 &quot;x 6&quot; di rosso per impronte dentarie. Le zecche sono strettamente posizionati insieme in gruppi di 5 tick. </li><li> Mettere una piccola striscia di nastro adesivo sopra l&#39;apparato boccale di tutte e 5 le zecche al fine di contenere ulteriormente, ma lasciando la maggior parte del corpo esposto in modo che il processo di iniezione può essere osservata dalla zecca iniettore (Figura 1). </li></ol> 2.4. Iniezione di zecche. <ol><li> Le zecche sarà iniettato nel quadrante in basso a destra della superficie ventrale della esoscheletro. </li><li> In primo luogo, forare un buco nella esoscheletro usando una siringa per insulina Monoject dotato di un ½ &quot;, 29 gauge (Figura 2a). </li><li> Iniettare zecche immediatamente con 0,2-0,5 ml di soluzione dsRNA (5 x 10 10 - 5 x 10 11 molecole per mL) con una misura siringa Hamilton con un pollice 1, 33 gauge con un 45 ° smussato punto (Figura 2b) . L&#39;ago deve essere inserito anche nella cavità tick per assicurare il posizionamento e la ritenzione del dsRNA. Alcuni fluido è probabile fuga dal sito di iniezione (figura 2c). Si deve prestare attenzione a non iniettare il zecche, che causerebbe la perdita di emolinfa e potrebbe causare la morte della zecca. </li><li> Pulire la siringa Hamilton dopo aver completato le iniezioni in ciascun gruppo sperimentale. prima di utilizzare per un altro gruppo sperimentale. Riempire la siringa prima da un bicchiere contenente acqua ossigenata al 3% e poi espellere in un contenitore dei rifiuti, e ripetere 15 volte. Riempire la siringa da un bicchiere contenente acqua sterile e poi espellere in un contenitore dei rifiuti, e ripetere 15 volte. Fare attenzione a non piegare lo stantuffo della siringa Hamilton perché, se piegato, il pistone non si muove senza problemi e rispondere al tocco gentile richiesta per l&#39;iniezione di zecche. </li></ol> 2.5. Trattamento di zecche dopo l&#39;iniezione. <ol><li> Sollevare il segno di spunta iniettato immediatamente dal doppio nastro adesivo con la pinza sottile e metterla in un contenitore di plastica di recupero (circa 6 &quot;x 6&quot; e circondata da nastro adesivo per evitare la fuga delle zecche). Le zecche saranno brevemente inattivo dopo l&#39;iniezione, ma dovrebbe presto cominciare a strisciare intorno al piatto. </li><li> Respiro di CO 2 sul zecche subito dopo metterli nel contenitore di recupero per aiutare ad attivare le zecche. Una volta che le zecche sono striscianti e attivo, la ferita iniezione guarirà rapidamente e saranno molto probabilmente sopravvivere. </li><li> Contare le zecche in base al numero di ogni gruppo sperimentale e metterli in un bicchiere di plastica marcato con un coperchio a tenuta montato. Zecche sostituzione deve essere iniettato per sostituire qualsiasi che ogni morto prima di iniettare il prossimo gruppo sperimentale. </li></ol> 2.6. Tick ​​azienda. <ol><li> Posizionare il zecche in una camera umida (12 ore di luce: 12 ore fotoperiodo buio a 22-25 ° C e 95% rispetto humidità) e tenere premuto per 1 giorno. </li><li> Zecche posto in tick-alimentare celle, una per ogni gruppo sperimentale, incollato a una pecora e permettere loro di alimentare con un numero uguale di uninjected zecche maschio o femmina (a seconda del sesso non è stato iniettato). Zecche femminile che si nutrono a sazietà, quelli che vengono rimossi dalle pecore dopo 10 giorni di alimentazione o quando le femmine di controllo sono caduto fuori l&#39;host sono raccolti e pesati. </li><li> Posizionare il zecche in cartoni, e tenere nella camera di umidità fino al completamento della deposizione delle uova. Valutare deposizione delle uova ponderando la massa di uova prodotte da tutte le zecche del gruppo. </li></ol> 2.7. Analisi del fenotipo tick dopo RNAi. <ol><li> Valutare tick fenotipo dopo l&#39;alimentazione, determinando il numero di zecche che sono sopravvissuti, il peso zecca, deposizione delle uova e la fertilità delle uova. Tuttavia, altre analisi può essere eseguita a seconda del gene bersaglio e gli obiettivi dello studio. </li></ol> 3. Analisi per confermare silenziamento genico mediante RT-PCR. <ol><li> Sezionare ghiandole salivari e coraggio da zecche singoli gruppi di controllo iniezione e iniezione di dsRNA dopo la poppata. </li><li> Estrarre l&#39;RNA totale da campioni di tessuto individuali. </li><li> Analizzare le trascrizioni gene bersaglio nei tessuti singole real-time RT-PCR e normalizzare i livelli di RNA contro le zecche rRNA 16S con il metodo genNorm (metodo DDCT come attuato da Bio-Rad iQ5 Standard Edition, versione 2.0). </li><li> Eseguire curve di dissociazione, alla fine della reazione per garantire che solo un amplicone è formato e che la denaturare ampliconi costantemente nella gamma stessa temperatura per ogni campione. </li><li> Confrontare i livelli di mRNA (normalizzato valori Ct) tra controllo iniezione e iniezione di dsRNA zecche utilizzando la Student `s t-test (p = 0,05). </li></ol> 4. Rappresentante dei risultati: Il protocollo qui descritto è stato utilizzato nel nostro laboratorio per RNAi in molte specie differenti zecche ixodid (Tabella 1). La quantità di dsRNA iniettato nel zecche varia con le dimensioni della zecca; specie di zecche più grande può ospitare un volume maggiore. Zecche controllo negativo deve essere iniettato con un dsRNA indipendenti. DsRNAs diversi come subolesin 14-19,22-25,27-32,34 e beta-actina 20,21 potrebbero essere utilizzati come controlli positivi. Si noti che è importante lavare la siringa tra i trattamenti per evitare la miscelazione di soluzioni dsRNA. Se il protocollo viene fatto correttamente, meno del 5% di mortalità dovrebbe essere ottenute con la procedura di iniezione dopo 24 ore. Un fenotipo tipico dopo knockdown gene nelle zecche è mostrato in figura 3 con un pannello di zecche iniettato con piscine di dsRNA in modo da schermo per tick antigeni protettivi. <table border="1"><tbody><tr><td> Tick ​​specie </td><td> dsRNA iniettato </td><td> Referenze </td></tr><tr><td> Ixodes scapularis </td><td> libreria di cDNA, subolesin, actina, nucleotidasi, NF-kB, akirin </td><td> 21, 22, 29, 30 </td></tr><tr><td> Dermacentor variabilis </td><td> subolesin, GST, ubiquitina, vATPase, selenoproteine ​​M e W2A, ematopoietiche staminali / progenitrici di proteine ​​come, subunità actina proteasoma 26S, ferritin1, varisin, akirin </td><td> 15, 19, 22, 24, 26, 30-32 </td></tr><tr><td> Dermacentor marginatus </td><td> subolesin </td><td> 22 </td></tr><tr><td> Amblyomma americanum </td><td> libreria di cDNA, subolesin, akirin </td><td> 17, 22, 30 </td></tr><tr><td> Amblyomma hebraeum </td><td> subolesin, voraxin </td><td> 28 </td></tr><tr><td> Rhipicephalus sanguineus </td><td> Rs86, subolesin </td><td> 22, 23 </td></tr><tr><td> Rhipicephalus Microplus </td><td> GST, ubiquitina, selenoproteina, Bm86, Bm91, subolesin, GI, GIII, EF1a, proteine ​​della seta flagelliform, von Willebrand fattore </td><td> 16, 18, 25, 27 </td></tr><tr><td> Rhipicephalus annulatus </td><td> ubiquitina, subolesin, EF1a, GIII </td><td> 16 </td></tr></tbody></table> Tabella 1. Tick specie in cui il protocollo RNAi è stato usato. <img src="/files/ftp_upload/2474/2474fig1.jpg" alt="Figura 1" /> Figura 1. Posizionamento delle zecche, lato ventrale, sulla doppio nastro adesivo aderito a un foglio di colore rosso per impronte dentarie. Le zecche sono inseriti in gruppi di 5, dopo il quale una piccola striscia di nastro adesivo è posta sopra l&#39;apparato boccale al fine di garantire ulteriormente le zecche, consentendo l&#39;iniettore di osservare il corpo della zecca durante l&#39;iniezione. <img src="/files/ftp_upload/2474/2474fig2.jpg" alt="Figura 2" /> Figura 2. La procedura di iniezione comprende (a) penetrante quadrante in basso a destra della zecca con un esoscheletro insulina syRinge con ago 29 gauge in modo da creare un sito di iniezione, (b) l&#39;iniezione immediata del dsRNA in questo sito utilizzando una siringa Hamilton con un ago 33 gauge, che (c) molto probabilmente si tradurrà in una perdita di emolinfa tick / fluidi. <img src="/files/ftp_upload/2474/2474fig3.jpg" alt="Figura 3" /> Figura 3. Un gruppo di zecca sei gruppi in cui è stato utilizzato per lo screening RNAi per gli antigeni tick di protezione Amblyomma americanum. I cambiamenti fenotipici in zecche possono essere visti se confrontato con il lato positivo subolesin RNAi di controllo e il controllo negativo dsRNA indipendenti. In questo esperimento l&#39;effetto di RNAi sulla mortalità zecca, pesi, e la deposizione di ogni gruppo sono stati analizzati statisticamente.

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Biol. 9, 42-42 (2009).</li><li>Zivkovic, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subolesin+expression+in+response+to+pathogen+infection+in+ticks.">Subolesin expression in response to pathogen infection in ticks.</a> BMC Immunol. 11, 7-7 (2010).</li><li>Karim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomics+tool:+gene+silencing+in+Ixodes+scapularis+eggs+and+nymphs+by+electroporated+dsRNA.">Functional genomics tool: gene silencing in Ixodes scapularis eggs and nymphs by electroporated dsRNA.</a> BMC Biotechnol. 10, 1-1 (2010).</li><li>Kocan, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transovarial+silencing+of+the+subolesin+gene+in+three-host+ixodid+tick+species+after+injection+of+replete+females+with+subolesin+dsRNA.">Transovarial silencing of the subolesin gene in three-host ixodid tick species after injection of replete females with subolesin dsRNA.</a> Parasitol. Res. 100, 1411-1415 (2007).</li><li>de la Fuente, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+tick-pathogen+interface+for+novel+control+strategies.">Targeting the tick-pathogen interface for novel control strategies.</a> Front. Biosci. 13, 6947-6956 (2008).</li><li>Kurscheid, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+a+tick+RNAi+pathway+by+comparative+genomics+and+reverse+genetics+screen+of+targets+with+known+loss-of-function+phenotypes+in+Drosophila.">Evidence of a tick RNAi pathway by comparative genomics and reverse genetics screen of targets with known loss-of-function phenotypes in Drosophila.</a> BMC Mol. Biol. 10, 26-26 (2009).</li></ol>

Nessun conflitto di interessi dichiarati.

Anche se altri metodi sono stati descritti per RNAi nelle zecche 14, 33, l&#39;iniezione di dsRNA qui descritto è il più utilizzato in entrambe le digiuno (Tabella 1) e nutriti zecche 16,25,34. RNAi ha dimostrato di essere un valido strumento per lo studio della funzione tick genica, la caratterizzazione del tic-patogeno interfaccia e lo screening e la caratterizzazione di antigeni tick di protezione 14,35. In particolare, la RNAi è diventato lo strumento più prezioso per l&#39;analisi funzionale nelle zecche 35. Metodologicamente, RNAi probabilmente evolverà in metodi più efficienti che possono permettere knockdown gene in un gran numero di individui. Il meccanismo di dsRNA indotta RNAi nelle zecche dovrebbe essere raffinato per contribuire ad una migliore comprensione e l&#39;utilizzo di questo approccio genetico in questa specie di 35,36. Il grado di off-target effetti della RNAi nelle zecche è anche una questione importante che deve essere pienamente affrontati 14,27. Infine, RNAi sarà molto probabilmente fornire contributi completa allo studio della regolazione genica e zecche biologia dei sistemi e il tic-patogeno interfaccia e possono avere un impatto sullo sviluppo di vaccini per controllare le infestazioni di zecche e la trasmissione di zecche patogeni.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Access RT-PCR system</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>A1250</td>
<td>&nbsp;</td>
<tr>
<td>Purelink PCR purification kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>K3100-02</td>
<td>&nbsp;</td>
<tr>
<td>Megascript RNAi kit</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>AM1626M</td>
<td>&nbsp;</td>
<tr>
<td>Red dental wax</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>72674</td>
<td>&nbsp;</td>
<tr>
<td>Plastic cups, 1.25 oz and lids</td>
<td>&nbsp;</td>
<td>Solo Cup Company, Urbana Ill.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fine forceps</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>Various</td>
<td>&nbsp;</td>
<tr>
<td>Insulin syringe</td>
<td>&nbsp;</td>
<td>Monoject</td>
<td>&nbsp;</td>
<td>Fitted with a &frac12;&#x201D;, 29 gauge needle</td>
</tr>
<tr>
<td>Hamilton syringe</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>701SN,33/.375&#x201D;/45DGR</td>
<td>Custom made</td>
</tr>
<tr>
<td>TriReagent</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>93289</td>
<td>&nbsp;</td>
<tr>
<td>iScript One-Step RT-PCR Kit with SYBR Green</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>170-8892</td>
<td>&nbsp;</td>
<tr>
<td>Real-time PCR detection system</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>Several</td>
<td>Please refer to <a href="http://www.bio-rad.com/">http://www.bio-rad.com/</a></td>
</tr>		</tbody>
		</table>
		</div>

rna interference in ticks

Le zecche sono ectoparassiti ematofagi obbligati degli animali selvatici e domestici e gli esseri umani, e sono considerati il secondo al mondo per le zanzare come vettori di malattie umane 1 e i vettori più importanti che riguardano l&#39;industria del bestiame in tutto il mondo 2. Le zecche sono classificate nella sottoclasse Acari, Parasitiformes ordine, sottordine Ixodida e sono distribuiti in tutto il mondo da Arctic alle regioni tropicali 3. Nonostante gli sforzi per controllare le infestazioni di zecche, ectoparassiti questi rimangono un serio problema per la salute umana e animale 4,5. Interferenza dell&#39;RNA (RNAi) 6 è un acido nucleico approccio inverso genetico che comporta interruzione di espressione genica per determinare la funzione del gene o il suo effetto su una via metabolica. Piccoli RNA interferenti (siRNA) sono le molecole effettrici della via RNAi che è iniziata da RNA a doppio filamento (dsRNA) e si traduce in un potente degradazione sequenza-specifica degli mRNA citoplasmatici contenenti la stessa sequenza come trigger dsRNA 7-9. Post-trascrizionale meccanismi di silenziamento genico iniziata da dsRNA sono stati scoperti in tutti gli eucarioti studiato finora, e RNAi è stata rapidamente sviluppata in una varietà di organismi come strumento per studi di genomica funzionale e altre applicazioni 10. RNAi è diventato il più utilizzato silenziamento genico tecnica zecche e altri organismi in cui approcci alternativi per la manipolazione genetica non sono disponibili o non sono affidabili 5,11. La caratterizzazione genetica delle zecche è stata limitata fino alla recente applicazione di RNAi 12,13. Nel poco tempo che RNAi è stato disponibile, ha dimostrato di essere uno strumento prezioso per studiare la funzione del gene tick, la caratterizzazione del tic-patogeno interfaccia e lo screening e la caratterizzazione di antigeni tick di protezione 14. Qui, un metodo per RNAi mediante iniezione di dsRNA in zecche digiuno è descritta. E &#39;probabile che la conoscenza acquisita da questo approccio sperimentale contribuirà notevolmente alla comprensione dei sistemi biologici di base e lo sviluppo di vaccini per controllare le infestazioni zecche e prevenire la trasmissione di zecche patogeni 15-19.

Watch this Scientific Journal Video about RNA Interference in Ticks at JoVE.com

RNA Interference in Ticks

Un metodo per l&#39;interferenza dell&#39;RNA (RNAi) mediante iniezione di dsRNA in zecche digiuno è descritta. RNAi è il più utilizzato silenziamento genico tecnica zecche dove l&#39;utilizzo di altri metodi di manipolazione genetica è stato limitato.

RNA interferenza nelle zecche

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

Research

JoVE Journal

Immunology and Infection

18.0K Views.  Oklahoma State University. Un metodo per l'interferenza dell'RNA (RNAi) mediante iniezione di dsRNA in zecche digiuno è descritta. RNAi è il più utilizzato silenziamento genico tecnica zecche dove l'utilizzo di altri metodi di manipolazione genetica è stato limitato.

Video: RNA Interference in Ticks

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery. 
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

Sviluppo di Cell-tipo specifico anti-HIV gp120 aptameri per la consegna di siRNA

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

Ricerca

Immunologia e infezioni

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Saliva, le ghiandole salivari, e Collection emolinfa dalle zecche Ixodes scapularis

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the attachment and blood feeding of Ixodes species ticks on mammalian hosts. In nature, this zoonotic bacterial pathogen may use a variety of reservoir hosts, but the white-footed mouse (Peromyscus leucopus) is the primary reservoir for larval and nymphal ticks in North America. Humans are incidental hosts most frequently infected with B. burgdorferi by the bite of ticks in the nymphal stage. B. burgdorferi adapts to its hosts throughout the enzootic cycle, so the ability to explore the functions of these spirochetes and their effects on mammalian hosts requires the use of tick feeding. In addition, the technique of xenodiagnosis (using the natural vector for detection and recovery of an infectious agent) has been useful in studies of cryptic infection. In order to obtain nymphal ticks that harbor B. burgdorferi, ticks are fed live spirochetes in culture through capillary tubes. Two animal models, mice and nonhuman primates, are most commonly used for Lyme disease studies involving tick feeding. We demonstrate the methods by which these ticks can be fed upon, and recovered from animals for either infection or xenodiagnosis.

Lyme disease is the most commonly-reported vector-borne disease in North America. The causative agent, Borrelia burgdorferi is a spirochete bacterium transmitted by Ixodid ticks. Transmission and detection of infection in animal models is optimized by the use of tick feeding, which we describe here.

Alimentazione di zecche su animali per trasmissione e Xenodiagnosis a Lyme Disease Research

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the  attachment and blood feeding of Ixodes species ticks on ...

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

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

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

Usando RNA-interferenza per Indagare sulla risposta immunitaria innata in mouse macrofagi

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

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

Nucleocapside Ricottura-Mediated elettroforesi (NAME) Assay permette la rapida identificazione del virus HIV-1 nucleocapside Inibitori

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

L&#39;applicazione di un sistema di espressione inducibile per studiare interferenza di fattori batterici di virulenza con segnalazione intracellulare

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

RNA Purificazione da nella cellula Grown<em&gt; Listeria monocytogenes</em&gt; In macrofagi

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated from na&#239;ve CD4+ T cells in vitro by activating them in the presence of the polarizing cytokines IL-1&#946;, IL-6, IL-23, and TGF&#946;. Th17 cells orchestrate immunity against extracellular bacteria and fungi, but their aberrant activity has also been associated with several autoimmune and inflammatory diseases. Th17 cells are identified by the chemokine receptor CCR6 and defined by their master transcription factor, ROR&#947;t, and characteristic effector cytokine, IL-17A. Optimized culture conditions for Th17 cell differentiation facilitate mechanistic studies of human T cell biology in a controlled environment. They also provide a setting for studying the importance of specific genes and gene expression programs through RNA interference or the introduction of microRNA (miRNA) mimics or inhibitors. This protocol provides an easy to use, reproducible, and highly efficient method for transient transfection of differentiating primary human Th17 cells with small RNAs using a next generation electroporation device.

Next generation electroporation is an efficient method for transfecting human Th17 cells with small RNAs to alter gene expression and cell behavior.

Piccolo RNA trasfezione in cellule primarie Th17 Human Next Generation elettroporazione

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

Identificazione di Coding e classi di RNA Non codificanti espressa in suina sangue intero

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

Formazione immagine confocal di RNA Double-Stranded e Pattern Recognition Receptors in infezione Virus RNA negativo

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...