Wir möchten Abbie Spinner an der California National Primate Research Center für ihre Unterstützung bei der Durchflusszytometrie danken. Diese Studie wurde durch Mittel aus dem National Institute of Allergy and Infectious Diseases (NIAID) der National Institutes of Health (NIH) AI073745, AI080799 und AI078183 unterstützt.

<ol><li> Vor dem Start des Protokolls ist es wichtig, festzustellen, dass die Mücke Zellen im Experiment verwendeten gesund sind und bei ~ 80% Konfluenz, wenn Anhänger (Abbildung 1). Overgrown oder kranken Zellen wird in geringer Transfektionseffizienz führen und inkonsistente Ergebnisse liefern. </li><li> Warm up alle Medien vor, in einem 28 ° C Wasserbad für 30 Minuten verwenden. <ol class="lalpha"><li> A. stephensi MSQ43 Zellen benötigen E5 Medium: MEM, Earle w / Glutamin, 5% hitzeinaktiviertem FCS, 0,2% D-Glucose, 1% Penicillin-Streptomycin Antibiotikum, und 1% nicht-essentiellen Aminosäuren. </li><li> A. gambiae SUA5B Zellen benötigen S2 Medium: Schneiders Drosophila Medium, 5% hitzeinaktiviertem FCS und 1% Penicillin-Streptomycin Antibiotikum </li></ol></li><li> Tauwetter und speichern Sie alle Transfektionsreagenz Materialien, Plasmide und Oligonukleotide auf dem Eis. </li><li> Alle Verfahren sollten in einer Gewebekultur Kapuze mit sterilen Technik durchgeführt werden. </li><li> Für adhärente Zellen (A. gambiae SUA5B, A. stephensi MSQ43) absaugen alten Medien, ohne die Zellen, fügen Sie eine äquivalente Menge an frischem Medium, das zu 28 wurde ° C erwärmt und dann abspülen Zellen vom Boden der Kolben mit einer Pipette. </li><li> Die Konzentration der Zellen zu bestimmen und angepasst, um eine endgültige Konzentration von 1x10 6 Zellen / ml. Die Zellen können bei Raumtemperatur aufbewahrt werden während der Vorbereitung Transfektionsreagenz Materialien. </li><li> 1 ml der Moskito-Zellen (1x10 6 Zellen / ml) in jede Vertiefung einer 6-Well-Platte. Richten Sie eine Platte für jede experimentelle Bedingung (siehe unten). Tabelle I ist ein Pipettierschema für eine allgemeine Transfektion. Tabelle II stellt fünf Bedingungen für eine erfolgreiche Mutagenese eines einzelnen Nukleotids mit BFP-spezifische LNA-ONs testen. Die ersten beiden, pGFP und pBFP, sind positive und negative Kontrollen. Die restlichen sind experimentelle Bedingungen pBFPs mit steigenden Konzentrationen von BFP-spezifische LNA-ons. In den Schritten 8 bis 12, fügen Sie entsprechende Volumina der Reagenzien entsprechend für jede Bedingung. </li><li> Transfer-Plasmid-DNA in ein steriles 1,5 ml Reaktionsgefäß und fügen entsprechende Volumen von EC-Puffer. <ol class="lalpha"><li> Für 1 ul Plasmid-DNA (1 pg / mL), fügen Sie 99 ul der EG-Puffer. </li><li> Für 1 ul pGFP (1 g / mL), fügen Sie 99 ul der EG-Puffer. </li><li> Für 1 ul pBFP (1 g / mL), fügen Sie 99 ul der EG-Puffer. </li><li> Für 1 ul pBFP (1 g / mL) und 1 ul ON (pg / mL), fügen Sie 98 ul der EG-Puffer. </li><li> Für 1 ul pBFP (1 g / mL) und 5 ul ON (1 g / mL), fügen Sie 94 ul der EG-Puffer. </li><li> Für 1 ul pBFP (1 g / mL) und 10 ul ON (1 g / mL), fügen Sie 89 ul der EG-Puffer. </li></ol></li><li> Dann langsam zugeben Enhancer pro 1 pg DNA verwendet. <ol class="lalpha"><li> Für 1 ul Plasmid-DNA, addieren Sie 8 ul Enhancer. </li><li> Für 1 ul pGFP, addieren Sie 8 ul Enhancer. </li><li> Für 1 ul pBFP, addieren Sie 8 ul Enhancer. </li><li> Für 1 ul pBFP und 1 ul ON, werden 16 ul Enhancer. </li><li> Für 1 ul pBFP und 5 ul ON, fügen Sie 48 ul Enhancer. </li><li> Für 1 ul pBFP und 10 ul ON, fügen Sie 88 ul Enhancer. </li></ol></li><li> Vortex DNA / Puffer / Enhancer Mischung für 1 sec und stellen Sie sicher, die gesamte Lösung ist an der Unterseite des Rohres. Dann bei Raumtemperatur Inkubation für 5 min. </li><li> Add Effectene Reagenz pro 1 pg Plasmid-DNA und Vortex für 10 Sekunden. Bestätigen Sie, dass die gesamte Lösung im unteren Bereich der Röhre und dann bei Raumtemperatur inkubieren für 10 min. <ol class="lalpha"><li> Für 1 ul Plasmid-DNA, mit 10 ul Effectene (Tabelle I). </li><li> Für 1 ul pGFP, mit 10 ul Effectene (Tabelle II). </li><li> Für 1 ul pBFP, mit 10 ul Effectene (Tabelle II). </li><li> Für 1 ul pBFP und 1 ul ON, mit 20 ul Effectene (Tabelle II). </li><li> Für 1 ul pBFP und 5 ul ON, mit 60 ul Effectene (Tabelle II). </li><li> Für 1 ul pBFP und 10 ul ON, fügen Sie 110 ul Effectene (Tabelle II). </li></ol></li><li> Bringen Sie jedes Röhrchen auf ein Endvolumen von 1000 ul, indem erwärmt Medien tropfenweise zu der DNA / Puffer / Enhancer / Effectene Mischung. DNA ausfallen kann an dieser Stelle, wenn die Medien zu schnell aufgenommen wird. Vorsichtig durch Auf-und Abpipettieren 3-4 mal zu mischen. <ol class="lalpha"><li> Für 1 ul Plasmid-DNA, fügen Sie 882 ul Medium (Tabelle I). </li><li> Für 1 ul pGFP, fügen Sie 882 ul Medium (Tabelle II). </li><li> Für 1 ul pBFP, fügen Sie 882 ul Medium (Tabelle II). </li><li> Für 1 ul pBFP und 1 ul ON, fügen Sie 864 ul Medium (Tabelle II). </li><li> Für 1 ul pBFP und 5 ul ON, fügen Sie 792 ul Medium (Tabelle II). </li><li> Für 1 ul pBFP und 10 ul ON, fügen Sie 702 ul Medium (Tabelle II). </li></ol></li><li> Next langsam 1 mL der entsprechenden DNA / Enhancer / Effectene Mischung in jede Vertiefung ein Tropfen auf einmal. </li><li> Schwenken Sie die Platte vorsichtig, um eine gleichmäßige Verteilung der Zellen und die Transfektion Mischung. INCUBATe-Zellen unter normalen Wachstumsbedingungen. </li><li> Einen Tag nach der Transfektion vorsichtig aspirieren aus Medien und langsam mit einem äquivalenten Volumen an frisch aufgewärmt Medien zu ersetzen. Es ist wichtig, nicht stören die Zellschichten an der Unterseite der Brunnen, wenn dies zu tun. </li><li> Zwei Tage nach der Transfektion untersucht positive Kontroll-Zellen mit pGFP unter Fluoreszenzmikroskop zu Transfektionseffizienz (Abbildung 2) zu bestimmen, transfiziert. </li><li> SUA5B Zellen wachsen sehr schnell und wird überwuchert. Vier Tage nach der Transfektion dünne dieser Zellen durch Absaugen aus den alten Medien und das Hinzufügen langsam ein äquivalentes Volumen an frisch aufgewärmt Medien. </li><li> Nach dem Hinzufügen neuer Medien in die Vertiefungen, trennen Sie alle Zellen aus dem Boden jeder Vertiefung durch Pipettieren auf und ab, werfen Sie die Hälfte des Volumens (1 ml) der Zelle Mischung geben und 1 ml frisches erwärmt Medien auf das Endvolumen auf 2 zu bringen mL. </li><li> Überprüfen Zellen täglich um sicherzustellen, dass sie gesund sind und nicht überwuchert, dünn wie nötig. </li><li> Lassen Sie die Zellen, die unter normalen Bedingungen für die fünf Minuten vor sieben Tagen nach der Transfektion wachsen, bevor sie zu sammeln für die Analyse. </li><li> In fünf Minuten vor sieben Tagen nach der Transfektion, sammeln die Zellen durch Absaugen der alten Medien in alle Brunnen und die Zugabe von 1 ml sterile Raumtemperatur PBS in jede Vertiefung. </li><li> Lösen Zellen vom Boden der gut-und Abpipettieren und Transfer Zellgemisch zu 5 ml Schnappdeckel Rohre und sofort analysieren Zellen über Durchflusszytometrie. </li></ol> Repräsentative Ergebnisse <img src="/files/ftp_upload/2355/2355fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Transfizierte gesunde A. stephensi MSQ43 (A) und A. gambiae SUA5B (B)-Zellen bei etwa 80% Konfluenz. <img src="/files/ftp_upload/2355/2355fig2.jpg" alt="Abbildung 2" /> Abbildung 2. A. stephensi MSQ43 (A) und A. gambiae SUA5B (B) Zellen mit positiven Kontrolle GFP-exprimierenden Plasmid transfiziert. <img src="/files/ftp_upload/2355/2355fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Durchflusszytometrie Daten bestätigt Umwandlung von BFP mit GFP über Mutagenese eines einzelnen Nukleotids nach der Transfektion mit BFP-spezifische gesperrt Nukleinsäure-modifizierte Oligonukleotide (LNA-ons). SUA5B Zellen wurden mit einem GFP-exprimierenden Plasmid (pGFP) als positive Kontrolle transfiziert, mit BFP exprimierenden Plasmid (pBFP) als negative Kontrolle, oder mit pBFP und steigenden Konzentrationen von BFP-spezifische LNA-ons. (A) Gating-Strategie für die Durchflusszytometrie Daten, die zeigen von links nach rechts: vorwärts im Vergleich side scatter von lebenden Zellen, Propidiumiodid (PI)-negativen Zellen und GFP-positiven Zellen 5 Tage nach der Transfektion. (B) Graph des Prozentsatzes der GFP-positiven Zellen in (A) nach der Transfektion mit pBFP und steigenden Konzentrationen von LNA-ONs gezeigt. <img src="/files/ftp_upload/2355/2355table1.jpg" alt="Tabelle 1" /> Tabelle I. Die Transfektion Bedingungen. <img src="/files/ftp_upload/2355/2355table2.jpg" alt="Tabelle 2" /> Tabelle II. Transfektion Bedingungen in Abbildung 1 verwendet.

<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>

Hier präsentieren wir ein in vitro Verfahren und Beweise für die gezielte Umwandlung von einem einzigen Nukleotid in einem episomalen Gen Ziel nach der Transfektion von LNA-ons in A. stephensi und A. gambiae Zellen. LNA-ON-vermittelte Gen-Konvertierung erfordert Induktion einer ortsspezifischen DNA-Reparatur-Reaktion, unsere Daten zeigen, dass Moskito-Zellen können diesen Mechanismus für die ortsspezifische Mutagenese zu unterstützen. Während die hier vorgestellte Methode auf Umwandlung ...

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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>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

Plasmodium Parasiten, der Erreger der Malaria, durch den Stich infizierter Anopheles-Mücken sich in mehr als 250 Millionen Neuinfektionen pro Jahr übertragen. Trotz jahrzehntelanger Forschung gibt es noch keinen Impfstoff gegen Malaria, die die Notwendigkeit für neue Bekämpfungsstrategien. Ein innovativer Ansatz ist die Verwendung von gentechnisch veränderten Mücken wirksam zu kontrollieren Malaria-Parasiten-Übertragung. Absichtliche Veränderungen der Zell-Signalwege in der Mücke, durch gezielte Mutagenese, haben sich Parasiten Entwicklung 1 zu regeln. Aus diesen Studien, können wir beginnen, um potenzielle Gen-Targets für die Transformation zu identifizieren. Durch gezielte Mutagenese wurde traditionell auf die homologe Rekombination zwischen einem Zielgen und ein großer DNA-Molekül verlassen. Allerdings ist der Aufbau und die Verwendung solcher komplexer DNA-Moleküle für die Erzeugung von stabil transformierten Zelllinien kostspielig, zeitaufwändig und oft ineffizient. Daher stellt eine Strategie mit gesperrten Nukleinsäure-modifizierte Oligonukleotide (LNA-ons) eine sinnvolle Alternative für die Einführung von künstlichen single nucleotide Substitutionen in episomalen und chromosomale DNA Gen-Targets (reviewed in 2). LNA-ON-vermittelte gezielte Mutagenese wurde verwendet, um Punktmutationen in Genen von Interesse in kultivierten Zellen von Hefe-und Mäuse 3,4 einzuführen. Wir zeigen hier, dass LNA-ONs verwendet werden, um ein einzelnes Nukleotid Änderung in einer transfizierten episomalen Ziel, das in einem Wechsel von blau fluoreszierenden Protein (BFP) zum Ausdruck grün fluoreszierende Protein (GFP)-Expression in beiden Anopheles gambiae und Anopheles stephensi Zellen führt einzuführen . Diese Umwandlung zeigt zum ersten Mal, dass eine wirksame Mutagenese von Zielgenen in Mücke-Zellen durch LNA-ons können vermittelt werden, und schlägt vor, dass diese Technik kann für Mutagenese von chromosomalen Ziele in vitro und 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

Oligonukleotide können die Besucher genutzt werden speziell Ersatz eines einzelnen Nukleotids von transfizierten Zielgene in beiden<em&gt; Anopheles gambiae</em&gt; Und<em&gt; Anopheles stephensi</em&gt; Zellen.

Transfektion und Mutagenese von Zielgenen in Mosquito-Zellen durch Locked Nucleic Acid-modifizierte Oligonukleotide

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 Aufrufe.  University of California, Davis. Oligonukleotide können die Besucher genutzt werden speziell Ersatz eines einzelnen Nukleotids von transfizierten Zielgene in beiden Anopheles gambiae Und Anopheles stephensi Zellen.

Serie: 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.

Identifizieren Dysregulated Gene von Kaposi-Sarkom-assoziierten Herpesvirus (KSHV) Induced

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 ...

Forschung

Immunologie und Infektion

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.

Antigen-spezifische In Vivo Killing-Assay mit CFSE markierten Zielzellen

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

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.

Optimiertes Protokoll für die effiziente Transfektion von dendritischen Zellen ohne Zell-Reifung

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.

Introducing Schubspannung in der Studie der bakteriellen Adhäsion

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.

Nach Cell-Schicksal<em&gt; E. coli</em&gt; Nach Infektion mit Phagen

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.

Die Beurteilung Somatische Hypermutation in Ramos B-Zellen nach Überexpression oder Knockdown von spezifischen Genen

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.

Locked Nucleic Acid Durchflusszytometrie-Fluoreszenz In situ Hybridisierung (LNA Flow-FISH): Eine Methode für die bakterielle RNA-Detektion

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

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.

Mit RNA-Interferenz, um die angeborene Immunantwort in Makrophagen der Maus Untersuchen

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

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.

Identifizierung neuer Gene, die mit Alginat-Produktion verbunden in<em&gt; Pseudomonas aeruginosa</em&gt; Mit Mini-<em&gt; Himar1</em&gt; Mariner Transposon-vermittelte Mutagenese

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.

Methods Article

Transfektion und Mutagenese von Zielgenen in Mosquito-Zellen durch Locked Nucleic Acid-modifizierte Oligonukleotide