Vorremmo ringraziare Abbie Spinner presso il Centro nazionale di ricerca sui primati della California per la sua assistenza con citometria a flusso. Questo studio è stato sostenuto da un finanziamento dell&#39;Istituto Nazionale di allergie e malattie infettive (NIAID) dei National Institutes of Health (NIH) AI073745, AI080799 e AI078183.

<ol><li> Prima di iniziare il protocollo, è importante stabilire che le cellule zanzara usati nell&#39;esperimento sono sani e alla confluenza ~ 80% se aderente (Figura 1). Cellule ricoperte o insalubri si tradurrà in bassa efficienza di trasfezione e daranno risultati inconsistenti. </li><li> Scaldare tutti i media prima di utilizzare in un bagno d&#39;acqua ° 28 C per 30 minuti. <ol class="lalpha"><li> A. stephensi MSQ43 cellule richiedono E5 medio: MEM, Earle w / glutammina, 5% FCS inattivato con il calore, lo 0,2% di D-glucosio, 1% di penicillina-streptomicina antibiotico, e 1% di aminoacidi non essenziali. </li><li> A. gambiae cellule SUA5B richiedono S2 medio: Schneider Drosophila medium, 5% FCS inattivato con il calore, e 1% di penicillina-streptomicina antibiotico </li></ol></li><li> Scongelare e conservare tutti i materiali reagenti di trasfezione, plasmidi e oligonucleotidi su ghiaccio. </li><li> Tutte le procedure devono essere effettuate in una cappa di coltura di tessuti con tecnica sterile. </li><li> Per le cellule aderenti (A. gambiae SUA5B, A. stephensi MSQ43) aspirare fuori i vecchi media senza disturbare le cellule, aggiungere un volume equivalente di mezzi freschi che è stato riscaldato a 28 ° C, e poi risciacquare le cellule dal fondo della beuta con una pipetta. </li><li> La concentrazione di cellule deve essere determinata e regolata per una concentrazione finale di 1x10 6 cellule / ml. Le celle possono essere conservati a temperatura ambiente durante la preparazione di materiali reagenti di trasfezione. </li><li> Aggiungere 1 ml di cellule di zanzara (1x10 6 cellule / ml) in ciascun pozzetto di un 6-pozzetti. Impostare un piatto per ogni condizione sperimentale (vedi sotto). Tabella I è un sistema di pipettaggio per una trasfezione generale. Tabella II illustra cinque condizioni per testare la mutagenesi di successo di un singolo nucleotide con BFP specifici LNA-ons. I primi due, pGFP e pBFP, sono controlli positivi e negativi, rispettivamente. I restanti sono le condizioni sperimentali di pBFPs con concentrazioni crescenti di BFP specifici LNA-ons. In passaggi da 8 a 12, aggiungere volumi adeguati di conseguenza per ogni reagente condizione. </li><li> Trasferimento di DNA plasmidico ad un tubo sterile 1,5 ml microcentrifuga e aggiungere volume adeguato di tampone CE. <ol class="lalpha"><li> Per 1 microlitri di DNA plasmidico (1 mg / mL), aggiungere 99 ml di tampone CE. </li><li> Per 1 pGFP microlitri (1μg / mL), aggiungere 99 ml di tampone CE. </li><li> Per 1 pBFP microlitri (1μg / mL), aggiungere 99 ml di tampone CE. </li><li> Per 1 pBFP microlitri (1μg / mL) e 1 ml ON (mg / mL), aggiungere 98 ml di tampone CE. </li><li> Per 1 pBFP microlitri (1μg / mL) e 5 microlitri ON (1μg / mL), aggiungere 94 ml di tampone CE. </li><li> Per 1 pBFP microlitri (1μg / mL) e 10 L ON (1μg / mL), aggiungere 89 ml di tampone CE. </li></ol></li><li> Poi aggiungere lentamente Enhancer per 1 mg di DNA utilizzato. <ol class="lalpha"><li> Per 1 microlitri di DNA plasmidico, aggiungere 8 microlitri Enhancer. </li><li> Per 1 pGFP microlitri, aggiungere 8 microlitri Enhancer. </li><li> Per 1 pBFP microlitri, aggiungere 8 microlitri Enhancer. </li><li> Per 1 pBFP microlitri e 1 ml ON, aggiungere 16 microlitri Enhancer. </li><li> Per 1 pBFP microlitri e 5 microlitri ON, aggiungere 48 microlitri Enhancer. </li><li> Per 1 pBFP microlitri e 10 L su ON, aggiungere 88 microlitri Enhancer. </li></ol></li><li> Vortex DNA / buffer / Enhancer miscela per 1 sec e assicurarsi che tutta la soluzione è nella parte inferiore del tubo. Incubare a temperatura ambiente per 5 min. </li><li> Aggiungere il reagente Effectene per 1 mg di DNA plasmidico e vortex per 10 sec. Verificare che tutta la soluzione è in fondo del tubo e poi incubare a temperatura ambiente per 10 min. <ol class="lalpha"><li> Per 1 microlitri di DNA plasmidico, Effectene aggiungere 10 microlitri (Tabella I). </li><li> Per 1 pGFP microlitri, Effectene aggiungere 10 microlitri (Tabella II). </li><li> Per 1 pBFP microlitri, Effectene aggiungere 10 microlitri (Tabella II). </li><li> Per 1 pBFP microlitri e 1 ml ON, Effectene aggiungere 20 microlitri (Tabella II). </li><li> Per 1 pBFP microlitri e 5 microlitri ON, aggiungere 60 microlitri Effectene (Tabella II). </li><li> Per 1 pBFP microlitri e 10 L su ON, aggiungere 110 microlitri Effectene (Tabella II). </li></ol></li><li> Portare ogni tubo ad un volume finale di 1000 ml con l&#39;aggiunta dei media riscaldato goccia a goccia al DNA / buffer / Enhancer / Effectene miscela. DNA può precipitare a questo punto se il supporto è aggiunto troppo velocemente. Mescolare delicatamente pipettando su e giù per 3-4 volte. <ol class="lalpha"><li> Per 1 microlitri di DNA plasmidico, aggiungere contenuti multimediali 882 microlitri (Tabella I). </li><li> Per 1 pGFP microlitri, aggiungere contenuti multimediali microlitri 882 (Tabella II). </li><li> Per 1 pBFP microlitri, aggiungere contenuti multimediali microlitri 882 (Tabella II). </li><li> Per 1 pBFP microlitri e 1 ml ON, aggiungere 864 microlitri dei media (Tabella II). </li><li> Per 1 pBFP microlitri e 5 microlitri ON, aggiungere 792 microlitri dei media (Tabella II). </li><li> Per 1 pBFP microlitri e 10 L su ON, aggiungere contenuti multimediali 702 microlitri (Tabella II). </li></ol></li><li> Prossimo aggiungere lentamente 1 mL di DNA appropriato / Enhancer / Effectene miscela in ciascun pozzetto una goccia alla volta. </li><li> Agitare delicatamente la piastra per assicurare una distribuzione uniforme delle cellule e la miscela di trasfezione. Incubatcellule e in normali condizioni colturali. </li><li> Un giorno dopo la transfezione, delicatamente aspirato fuori media e lentamente sostituire con un volume equivalente di media appena riscaldato. E &#39;importante non disturbare gli strati di cellule al fondo dei pozzetti durante questa operazione. </li><li> Due giorni dopo la transfezione esaminare le cellule di controllo positivo transfettate con pGFP al microscopio a fluorescenza per determinare l&#39;efficienza di trasfezione (Figura 2). </li><li> SUA5B cellule crescono molto rapidamente e sarà invaso. Quattro giorni dopo la transfezione sottile queste cellule aspirando i vecchi media e aggiungendo lentamente un volume equivalente di media appena riscaldato. </li><li> Dopo l&#39;aggiunta di nuovi media per i pozzi, staccare tutte le cellule dal fondo di ogni bene pipeting su e giù, scartare la metà del volume (1 mL) della miscela di cellule e aggiungere 1 ml di fresca mezzi riscaldato per portare il volume finale a 2 mL. </li><li> Controllare le cellule al giorno per assicurarsi che siano sani e non troppo lunghi, sottili, se necessario. </li><li> Permettono alle cellule di crescere in condizioni normali per 5-7 dopo trasfezione giorni prima della loro raccolta per l&#39;analisi. </li><li> Alle 06:55 dopo trasfezione giorni, raccogliere le cellule aspirando i vecchi media in tutti i pozzi e l&#39;aggiunta di 1 ml di PBS sterile a temperatura ambiente in ogni pozzetto. </li><li> Staccare le cellule dal fondo del pozzo pipettando su e giù e il trasferimento miscela di cellule a 5 tubi ml tappo a scatto e subito analizzare le cellule mediante citometria a flusso. </li></ol> Rappresentante Risultati <img src="/files/ftp_upload/2355/2355fig1.jpg" alt="Figura 1" /> Figura 1. Untransfected A. sano stephensi MSQ43 (A) e A. gambiae SUA5B (B), le cellule a circa 80% confluenza. <img src="/files/ftp_upload/2355/2355fig2.jpg" alt="Figura 2" /> Figura 2. A. stephensi MSQ43 (A) e A. gambiae SUA5B (B) cellule trasfettate con GFP controllo positivo che esprime plasmide. <img src="/files/ftp_upload/2355/2355fig3.jpg" alt="Figura 3" /> Figura 3. Citometria a flusso di dati a conferma di conversione di BFP per GFP attraverso mutagenesi di un singolo nucleotide dopo trasfezione con BFP specifici bloccati acido nucleico modificati oligonucleotidi (LNA-ons). SUA5B cellule sono state transfettate con un plasmide che esprime GFP (pGFP) come controllo positivo, con un plasmide che esprime BFP (pBFP) come controllo negativo, o con pBFP e concentrazioni crescenti di BFP specifici LNA-ons. (A) strategia di gating per i dati di citometria a flusso che mostrano da sinistra a destra: scatter in avanti rispetto a lato di cellule vive, propidio ioduro (PI), le cellule negative, e le cellule GFP positive cinque giorni dopo la transfezione. (B) Grafico della percentuale di cellule GFP positive mostrato in (A) dopo trasfezione con pBFP e concentrazioni crescenti di LNA-ons. <img src="/files/ftp_upload/2355/2355table1.jpg" alt="Tabella 1" /> Tabella I. Transfection condizioni. <img src="/files/ftp_upload/2355/2355table2.jpg" alt="Tabella 2" /> Tabella II. Condizioni Transfection utilizzato nella Figura 1.

<ol>
	<li>Corby-Harris, V. D. A., Watkins de Jong, L., Pakpour, N., Antonova, Y., Ziegler, R., Ramberg, F., Lewis, E. E., Brown, J. M., Luckhart, S. L., Riehle, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+strategy+for+controlling+malaria+transmission+in+the+mosquito+Anopheles+stephensi.">A novel strategy for controlling malaria transmission in the mosquito Anopheles stephensi.</a> PLOS Pathogens. , (2010).</li><li>Braasch, D. A., Corey, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locked+nucleic+acid+(LNA):+fine-tuning+the+recognition+of+DNA+and+RNA.">Locked nucleic acid (LNA): fine-tuning the recognition of DNA and RNA.</a> Chem Biol. 8, 1-7 (2001).</li><li>Andrieu-Soler, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+transmission+of+targeted+gene+modification+using+single-stranded+oligonucleotides+with+flanking+LNAs.">Stable transmission of targeted gene modification using single-stranded oligonucleotides with flanking LNAs.</a> Nucleic Acids Res. 33, 3733-3742 (2005).</li><li>Parekh-Olmedo, H., Drury, M., Kmiec, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+nucleotide+exchange+in+Saccharomyces+cerevisiae+directed+by+short+oligonucleotides+containing+locked+nucleic+acids.">Targeted nucleotide exchange in Saccharomyces cerevisiae directed by short oligonucleotides containing locked nucleic acids.</a> Chem Biol. 9, 1073-1084 (2002).</li><li>Rhee, W. J., Santangelo, P. J., Jo, H., Bao, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+accessibility+and+signal+specificity+in+live-cell+detection+of+BMP-4+mRNA+using+molecular+beacons.">Target accessibility and signal specificity in live-cell detection of BMP-4 mRNA using molecular beacons.</a> Nucleic Acids Res. 36, e30-e30 (2008).</li><li>Marras, S. A., Kramer, F. R., Tyagi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotyping+SNPs+with+molecular+beacons.">Genotyping SNPs with molecular beacons.</a> Methods Mol Biol. 212, 111-128 (2003).</li><li>Tyagi, S., Bratu, D. P., Kramer, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+molecular+beacons+for+allele+discrimination.">Multicolor molecular beacons for allele discrimination.</a> Nat Biotechnol. 16, 49-53 (1998).</li><li>Blandin, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+genetic+basis+of+resistance+to+malaria+parasites+in+Anopheles+gambiae.">Dissecting the genetic basis of resistance to malaria parasites in Anopheles gambiae.</a> Science. 326, 147-150 (2009).</li><li>Niare, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+loci+affecting+resistance+to+human+malaria+parasites+in+a+West+African+mosquito+vector+population.">Genetic loci affecting resistance to human malaria parasites in a West African mosquito vector population.</a> Science. 298, 213-216 (2002).</li><li>Riehle, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+malaria+infection+in+Anopheles+gambiae+is+regulated+by+a+single+genomic+control+region.">Natural malaria infection in Anopheles gambiae is regulated by a single genomic control region.</a> Science. 312, 577-579 (2006).</li></ol>

Nessun conflitto di interessi dichiarati.

Qui vi presentiamo un metodo in vitro e prove per la conversione mirata di un singolo nucleotide in un gene bersaglio episomiale dopo la transfezione di LNA-ons in A. stephensi e A. gambiae cellule. LNA-ON-mediata conversione genica richiede l&#39;induzione di un sito specifico per la risposta di riparazione del DNA, i nostri dati indicano che le cellule di zanzare in grado di supportare questo meccanismo di mutagenesi sito-specifici. Mentre il metodo qui presentato è basato sulla conversione...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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>Effectene transfection reagent</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>301425</td>
<td>&nbsp;</td>
<tr>
<td>GFP control plasmid (pcDNA6.2-GW/EmGFP-miR-neg)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>K4935-00</td>
<td>&nbsp;</td>
<tr>
<td>BFP plasmid (pcDNA5/FRT/TO)</td>
<td>&nbsp;</td>
<td>Gift from Dr. Concordet3</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>LNA-ONs </td>
<td>&nbsp;</td>
<td>Gift from Dr. Concordet3</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>MEM, Earle's w/ glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11095</td>
<td>&nbsp;</td>
<tr>
<td>Fetal calf serum (FCS)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16000</td>
<td>&nbsp;</td>
<tr>
<td>Schneider&#x2019;s Drosophila medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11730</td>
<td>&nbsp;</td>
<tr>
<td>Non-essential amino acids</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11140</td>
<td>&nbsp;</td>
<tr>
<td>10% D-glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G5767</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-streptomycin antibiotic</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>6-well culture plates</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>3516</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

transfection and mutagenesis of target genes in mosquito cells by locked nucleic acid-modified oligonucleotides

Parassiti Plasmodium, l&#39;agente eziologico della malaria, sono trasmessi attraverso la puntura di zanzare anofele con conseguente oltre 250 milioni di nuove infezioni ogni anno. Nonostante decenni di ricerca, non esiste ancora un vaccino contro la malaria, evidenziando la necessità di strategie di controllo romanzo. Un approccio innovativo è l&#39;uso di zanzare geneticamente modificati per il controllo dei parassiti in modo efficace la trasmissione della malaria. Alterazioni deliberate delle vie di segnalazione cellulare nella zanzara, tramite mutagenesi mirata, sono stati trovati a regolare parassita sviluppo 1. Da questi studi, possiamo cominciare a identificare gli obiettivi gene potenziale di trasformazione. Mutagenesi mirata è tradizionalmente invocata la ricombinazione omologa tra un gene target e una molecola di DNA di grandi dimensioni. Tuttavia, la costruzione e l&#39;uso di tali molecole di DNA complesse per la generazione di linee cellulari stabilmente trasformate è costoso, richiede tempo e spesso inefficiente. Pertanto, utilizzando una strategia chiuso acido nucleico modificati oligonucleotidi (LNA-ons) fornisce una valida alternativa per l&#39;introduzione artificiale sostituzioni di singoli nucleotidi in obiettivi DNA episomiale e cromosomiche gene (recensione in 2). LNA-ON-mediata mutagenesi previsioni è stato utilizzato per introdurre mutazioni puntiformi nei geni di interesse in cellule in coltura sia di lieviti e topi 3,4. Mostriamo qui che LNA-on può essere utilizzato per introdurre un cambiamento singolo nucleotide in un obiettivo transfettate episomiale che si traduce in un passaggio dal blu proteina fluorescente (BFP) espressione di proteina fluorescente verde (GFP) espressione in entrambe le Anopheles gambiae e Anopheles cellule stephensi . Questa conversione dimostra per la prima volta che la mutagenesi efficace di geni bersaglio in cellule di zanzara può essere mediata da LNA-on e suggerisce che questa tecnica può essere applicabile a mutagenesi di obiettivi cromosomiche in vitro e in vivo.

Watch this Scientific Journal Video about Transfection and Mutagenesis of Target Genes in Mosquito Cells by Locked Nucleic Acid-modified Oligonucleotides at JoVE.com

Transfection and Mutagenesis of Target Genes in Mosquito Cells by Locked Nucleic Acid-modified Oligonucleotides

Oligonucleotidi possono essere utilizzati per il sito specifico sostituire un singolo nucleotide dei geni bersaglio transfettate in entrambe le<em&gt; Anopheles gambiae</em&gt; E<em&gt; Anopheles stephensi</em&gt; Cellule.

Trasfezione e Mutagenesi di geni bersaglio in cellule Mosquito da Locked Nucleic Acid modificati Oligonucleotidi

Plasmodium parasites, the causative agent of malaria, are transmitted through the bites of infected Anopheles mosquitoes resulting in over 250 million new ...

transfection-mutagenesis-target-genes-mosquito-cells-locked-nucleic

Research

JoVE Journal

Immunology and Infection

10.3K Views.  University of California, Davis. Oligonucleotidi possono essere utilizzati per il sito specifico sostituire un singolo nucleotide dei geni bersaglio transfettate in entrambe le Anopheles gambiae E Anopheles stephensi Cellule.

Video: Transfection and Mutagenesis of Target Genes in Mosquito Cells by Locked Nucleic Acid-modified Oligonucleotides

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative tumor of vascular endothelial cells and produces rare B cell lymphoproliferative diseases in the form of pleural effusion lymphomas (PEL) and some forms of multicentric Castleman's disease.3-5 Only 1-5% of cells in KS lesions actively support lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent associated with KS, and it is clear that cellular factors must interact with viral factors in the process of oncogenesis and tumor progression.6,7 Identifying novel host-factor determinants which contribute to KS pathology is essential for developing prognostic markers for tumor progression and metastasis as well as for developing novel therapeutics for the treatment of KS.8 The accompanying video details the methods we use to identify host cell gene expression programs altered in dermal microvascular endothelial cells (DMVEC) after KSHV infection and in KS tumor tissue.9 Once dysregulated genes are identified by microarray analysis, changes in protein expression are confirmed by immunoblot and dual labeled immunofluorescence. Changes in transcriptional expression of dysregulated genes are confirmed in vitro by quantitative real-time polymerase chain reaction (qRT-PCR). Validation of in vitro findings using archival KS tumor tissue is also performed by dual labeled immunochemistry and tissue microarrays.8,10 Our approach to identifying dysregulated genes in the KS tumor tissue microenvironment will allow the development of in vitro and subsequently in vivo model systems for discovery and evaluation of potential novel therapeutic for the treatment of KS.

Identifying Dysregulated Genes Induced by Kaposi&#039;s Sarcoma-associated Herpesvirus KSHV

Host cell factors play a critical role in the establishment and maintenance of Kaposi's sarcoma (KS). We outline methods to identify host cell factors altered in KSHV-infected DMVEC cells, and in KS tumor tissue. Cellular genes altered by virus will serve as potential target(s) for novel therapeutics.

Identificare i geni disregolazione indotta da sarcoma di Kaposi associato Herpesvirus (KSHV)

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative ...

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Immunologia e infezioni

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells4-6. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing7,8. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities9. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.

Antigene specifico In Vivo Analisi Uccidere utilizzando cellule bersaglio CFSE etichetta

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

Preparazione e utilizzo di HIV-1 CD4 infetti primaria + T-Cells come cellule bersaglio nei test di cellule Natural Killer citotossici

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

Protocollo ottimizzato per Transfection efficiente delle cellule dendritiche, senza la maturazione delle cellule

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

L&#39;introduzione di shear stress nello studio di adesione batterica

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

A seguito di Cell-destino<em&gt; E. coli</em&gt; Dopo l&#39;infezione da fago lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes

B cells start their life with low affinity antibodies generated by V(D)J recombination. However, upon detecting a pathogen, the variable (V) region of an immunoglobulin (Ig) gene is mutated approximately 100,000-fold more than the rest of the genome through somatic hypermutation (SHM), resulting in high affinity antibodies1,2. In addition, class switch recombination (CSR) produces antibodies with different effector functions depending on the kind of immune response that is needed for a particular pathogen. Both CSR and SHM are initiated by activation-induced cytidine deaminase (AID), which deaminates cytosine residues in DNA to produce uracils. These uracils are processed by error-prone forms of repair pathways, eventually leading to mutations and recombination1-3.
Our current understanding of the molecular details of SHM and CSR come from a combination of studies in mice, primary cells, cell lines, and cell-free experiments. Mouse models remain the gold standard with genetic knockouts showing critical roles for many repair factors (e.g. Ung, Msh2, Msh6, Exo1, and polymerase &eta;)4-10. However, not all genes are amenable for knockout studies. For example, knockouts of several double-strand break repair proteins are embryonically lethal or impair B-cell development11-14. Moreover, sometimes the specific function of a protein in SHM or CSR may be masked by more global defects caused by the knockout. In addition, since experiments in mice can be lengthy, altering expression of individual genes in cell lines has become an increasingly popular first step to identifying and characterizing candidate genes15-18.
Ramos &#x2013; a Burkitt lymphoma cell line that constitutively undergoes SHM &#x2013; has been a popular cell-line model to study SHM18-24. One advantage of Ramos cells is that they have a built-in convenient semi-quantitative measure of SHM. Wild type cells express IgM and, as they pick up mutations, some of the mutations knock out IgM expression. Therefore, assaying IgM loss by fluorescence-activated cell scanning (FACS) provides a quick read-out for the level of SHM. A more quantitative measurement of SHM can be obtained by directly sequencing the antibody genes. 
Since Ramos cells are difficult to transfect, we produce stable derivatives that have increased or lowered expression of an individual gene by infecting cells with retroviral or lentiviral constructs that contain either an overexpression cassette or a short hairpin RNA (shRNA), respectively. Here, we describe how we infect Ramos cells and then use these cells to investigate the role of specific genes on SHM (Figure 1).

We describe how to perform retroviral or lentiviral infections of overexpression or shRNA-containing constructs in the human Ramos B-cell line and how to measure somatic hypermutation in these cells.

Valutare mutazione somatica nel Ramos cellule B dopo iperespressione o Knockdown di specifici geni

B cells start their life with low affinity antibodies generated by V(D)J recombination.  However, upon detecting a pathogen, the variable (V) region of an ...

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6.
Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5.
Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

A novel high-throughput method is described that enables the detection and relative quantitation of small RNA and mRNA expression from single bacterial cells using locked nucleic acid probes and flow cytometry-fluorescence in situ hybridization.

Chiuso flusso degli acidi nucleici Citometria fluorescenza In situ Ibridazione (LNA flow-FISH): un metodo per la rilevazione dei batteri piccoli RNA

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular ...

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial pathogen which establishes chronic pulmonary infections in patients with cystic fibrosis (CF). The overproduction of a capsular polysaccharide called alginate, also known as mucoidy, promotes the formation of mucoid biofilms which are more resistant than planktonic cells to antibiotic chemotherapy and host defenses. Additionally, the conversion from the nonmucoid to mucoid phenotype is a clinical marker for the onset of chronic infection in CF. Alginate overproduction by P. aeruginosa is an endergonic process which heavily taxes cellular energy. Therefore, alginate production is highly regulated in P. aeruginosa. To better understand alginate regulation, we describe a protocol using the mini-himar1 transposon mutagenesis for the identification of novel alginate regulators in a prototypic strain PAO1. The procedure consists of two basic steps. First, we transferred the mini-himar1 transposon (pFAC) from host E. coli SM10/&#955;pir into recipient P. aeruginosa PAO1 via biparental conjugation to create a high-density insertion mutant library, which were selected on Pseudomonas isolation agar plates supplemented with gentamycin. Secondly, we screened and isolated the mucoid colonies to map the insertion site through inverse PCR using DNA primers pointing outward from the gentamycin cassette and DNA sequencing. Using this protocol, we have identified two novel alginate regulators, mucE (PA4033) and kinB (PA5484), in strain PAO1 with a wild-type mucA encoding the anti-sigma factor MucA for the master alginate regulator AlgU (AlgT, &#963;22). This high-throughput mutagenesis protocol can be modified for the identification of other virulence-related genes causing change in colony morphology.

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Here we describe a protocol using the mini-himar1 mariner transposon-mediated mutagenesis for generating a high-density insertion mutant library to screen, isolate and identify novel alginate regulators in the prototypic Pseudomonas aeruginosa strain PAO1.

Identificazione di geni novelli associati con alginato di produzione in<em&gt; Pseudomonas aeruginosa</em&gt; Utilizzo Mini-<em&gt; Himar1</em&gt; Mutagenesi Mariner Transposon-mediata

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.