Wir möchten Ken Nealson für seine Einsicht und Beratung während des Manuskripts Prozess danken.

1. Herstellung von Virussuspensionen Vor Isolierung einzelner Virionen über Durchflusszytometrie, bereiten fixierten Virussuspensionen. <ol><li> Isolieren Viruspartikel mit vorher etablierten Protokollen sicherstellen endgültigen Virussuspension wird in 0,1 enthielt um filtriert TE (Tris-EDTA, pH 8). Wir empfehlen die Verwendung Tangentialflussfiltration (TFF) nähert 6 obwohl andere Methoden entwickelt wurden, die Verwendung chemischer Flockung 7. </li><li> Aufzählen Viruspartikel mit etablierten Protokollen, z. B. mit Epifluoreszenz 8-10 oder Durchflusszytometrie 11,12. </li><li> Aliquot viralen Suspension in zwei Eppendorf-Röhrchen (Endvolumen wird empfohlen, bis 1000 ul sein). </li></ol> 2. Durchflusszytometrie <ol><li> Um Viren mit dem BD FACSAria II Durchflusszytometer mit einem benutzerdefinierten Forward Scatter PMT (FSC PMT) ausgestattet sortieren, verdünnen Viruspartikel in 0,1 um filtriert TE, pH 8.0 zu einem geeigneten Titer für eine Veranstaltung von 200 Veranstaltungen sec -1. </li><li> Schwellenwerte, um das Signal-zu-Rausch-Rationen zu maximieren, zB. für eine gemischte Montage mit T4 und Lambda-Phagen-Partikel die folgende empfohlen: FSC PMT bei 1.000 und bei 200 SSC. </li><li> Run 100 ul &quot;blank&quot; Proben, die nur 1) 0,1 um filtriert und TE 2) 0,1 um filtriert und TE SybrGreen1 bei 1e-4 Verdünnung Lager (10.000 X). </li><li> Führen Sie ein kleines Aliquot (100 ul wird empfohlen) von ungefärbten viralen Suspension Hintergrund zu bewerten, durch die virale gefärbten Suspension (SybrGreen1 bei 1e-4 Verdünnung stock), virale Partikel zu insgesamt 5.000 Veranstaltungen pro gefolgt. Dies ist, um die Konzentration zu überprüfen und anzupassen Weichen, falls erforderlich, vor sort. Wir empfehlen eine enge Tor im Zentrum von Viruspartikeln. Auch zur Erzeugung von Toren nutzen wir die SYBR Green und FSC PMT Grundstück. </li><li> In 5 ul 1% niedrigem Schmelzpunkt (LMP) Agarose (in TBE-Puffer), abgekühlt auf 37 ° C zu jedemgut auf einem Polytetrafluorethylen (PTFE) Objektträger (Electron Microscopy Sciences). </li><li> Laden Sie die PTFE Objektträger in Durchflusszytometer sortiert viralen Partikel zu erfassen. </li><li> Beginnen Art von SYBR grün gefärbten Viruspartikel direkt auf PTFE Folie mit LMP-Agarose. Wir empfehlen Sortierung 10 oder mehr Veranstaltungen (Viruspartikel) in einigen Brunnen in der Erfassung der Tiefe der Viren während der konfokalen Laser-Scanning-Mikroskopie (CLSM) unterstützen. </li><li> Bei Sortierung abgeschlossen ist, entfernen Folie aus der Durchflusszytometer und mit 5 ul mehr LMP Agarose abgekühlt auf 37 ° C in jede Vertiefung, damit die Einbettung der Viruspartikel (n). </li></ol> 3. Visualizing Einzel Virionen mit konfokale Mikroskopie Wenn ein einzelnes Virion erwünscht ist, dann Bestimmen, ob jeder Vertiefung enthält eine einzelne Virus unumgänglich ist und CLSM ist notwendig, um die eingebettete Virion (s) in 3D um sicherzustellen, dass nur ein einzelnes Teilchen vorhanden ist und visualisieren mehrere Virensind nicht auf der jeweils anderen gestapelt. <ol><li> Stellen Sie das Mikroskop Laser der Wellenlänge 488 nm Anregung. </li><li> Bild die eingebetteten Viruspartikeln mit einem 63X langen Arbeitsabstand Ziel. </li></ol> 4. Whole Genome Amplification <ol><li> Sobald Vertiefungen mit einzelnen Viren mit CLSM identifiziert, führen Amplifikation des gesamten Genoms mit mehreren Displacement Amplification (MDA) in der Agarose (in situ). </li><li> Um die Wahrscheinlichkeit der Einführung von DNA-Kontaminationen zu verringern, nehmen Sie die gewünschten Agarose &#39;Perlen&#39; aus der Folie und in eine sterile PCR-Röhrchen. Verwenden Sie ein Paar sterile Pinzette und / oder einer Rasierklinge für diesen Schritt. </li><li> Lyse der viralen Partikel in situ unter Verwendung von Wärme (94 ° C) für 3 min in der Hitze Block von einem Thermocycler. </li><li> Führen Sie den MDA Reaktion folgenden Empfehlungen des Herstellers (GenomiPhi HY Kit, GE Healthcare) mit den folgenden Modifikationen <ol><li> Reduzieren Sie die Lautstärke der Probe und der Reaktion buffers auf 11,25 ul und Inkubation bei 30 ° C für maximal 2 Stunden, um unspezifische Amplifikation zu reduzieren. </li></ol></li><li> Reinigen Sie die amplifizierte genomische Material durch die Übertragung von genomischer DNA (gDNA) zu einem 1,7 ml Eppendorf-Röhrchen und fügen Sie 1/10 Volumen 3 M Natriumacetat (NaOAc) in TBE-Puffer (gleichen Puffer in LMP-Agarose Zubereitung verwendet). </li><li> Man gibt die Probe (n) bei 55 ° C für 10 min auflösen Agarose. </li><li> Bewegen Rohr (s) bis 42 ° C um die Temperatur zu reduzieren, bevor Zugabe des Enzyms. </li><li> In 2 Einheiten β-Agarase in jedes Röhrchen und Inkubation für 2 Stunden. </li><li> 1 Volumen eines Puffers-gesättigtem Phenol-Lösung mit dem Rohr, kräftig mischen und zentrifugieren bei 3.500 xg für 15 min. Überstand die DNA zu einem neuen 1,7 ml Eppendorf-Röhrchen. </li><li> In einem Volumen von 100% Isopropanol und 1 ul GlycoBlue. Gut mischen durch Umdrehen des Röhrchens. </li><li> Zentrifuge bei 28.000 xg bei 4 ° C für 60 min. Dekantieren Isopropanol was sicher nicht um das Pellet zu stören. In 150 ul 70% ethanol in jedes Röhrchen. Spin bei 28.000 xg und RT für 10 min. Dekantiert das Ethanol, trockene Luft und das DNA-Pellet resuspendieren die DNA in einem geeigneten Volumen TE-Puffer. </li><li> Wir empfehlen die Schritte 4.4-4.9 mit den folgenden Modifikationen. Einrichten 3-5 Replikat MDA Reaktionen und reduzieren Inkubation bei 30 ° C bis 1 Stunde. Pool-Proben nach der Reinigung und Niederschlag mit 95-100% Ethanol gewünschte Konzentration zu erhalten. </li></ol> 5. Repräsentative Ergebnisse Abbildung 1 fasst die SVG Prozess. Diese Methoden verwenden Durchflusszytometrie, um virale Partikel auf PTFE Objektträger mit Agarose einzelne Virionen aus einer gemischten Versammlung von MDA gefolgt, um Mengen von gDNA, die ausreichend sind für die Sequenzierung zu erhalten isolieren sortieren. Als Proof-of-concept, wurden Bakteriophagen Lambda und T4 gemischt und der SVG 11 Verfahren. Sobald Virionen wurden gefangen genommen und in Agarose eingebettet CLSM was zur Visualisierung und bestätigen, dass ein einzelnes Virion erhalten wurde;. Ergebnisse sind in Abbildung 2 gezeigt Sobald Brunnen mit einzelnen Virionen identifiziert wurden, wurden sie für MDA von gDNA ausgewählt. Wir haben dann verwendet Multiplex-PCR spezifisch für T4 und Lambda die Virion isoliert, Abbildung 3. Identifizieren Eine isolierte Phagen Lambda wurde für Genom-Sequenzierung ausgewählt. Genom-Sequenzierung wurde unter Verwendung von 454-Titanium-Technologie und Sequenzierung gelesen wurden einer Zuordnung Verweis auf das Niveau der Berichterstattung unter Verwendung SVG identifizieren. Abbildung 4 zeigt, dass fast die gesamte Lambda-Genom gewonnen wurde (mit Ausnahme der ersten 5 bp). <img alt="Abbildung 1" src="/files/ftp_upload/3899/3899fig1.jpg" /> Abbildung 1. SVG-Methodik. Viral Suspensionen, die eine gemischte Montage auf einzelne Viruspartikel über Durchflusszytometrie, die dann s reduziertorted auf einzelne Agarose &quot;Perlen&quot;. Das Virus wird im Agarose-Kügelchen durch Überlagerung mit einer zusätzlichen Schicht von Agarose Beitrag durchflusszytometrische Sortierung eingebettet. Schließlich wird Amplifikation des gesamten Genoms in situ durchgeführt. <img alt="Abbildung 2" src="/files/ftp_upload/3899/3899fig2.jpg" /> Abbildung 2. Die konfokale Laser-Scanning-Aufnahme einer sortierten Viruspartikel in einer Agarosekugel eingebettet. A) Dreidimensionale Rekonstruktion eines SYBR Green I gefärbten Viruspartikel innerhalb eines Agarosekugel. Einschub: höherer Vergrößerung des isolierten Viruspartikel B) Profil Diagramm der relativen Fluoreszenz der gefärbten einzigen Viruspartikel innerhalb eines Agarosekugel.. <img alt="Abbildung 3" src="/files/ftp_upload/3899/3899fig3.jpg" /> Abbildung 3. Bakteriophagen Identifizierung mittels PCR. A) &lt;/ Strong&gt; Multiplex PCR unter Verwendung von T4 und Lambda-spezifische Primer zu Genotyp Lanes: 1. TrackIt 1 kb zuzüglich Leiter (Invitrogen), 2. Lambda Integrase (750 bp), 3. T4 Hauptcapsid Protein (1.050 bp), 4. . Mix von Lambda Integrase und T4 Hauptcapsid Protein B) Nachfolgende Lambda spezifischen PCR mit zusätzlichen Loci weiter bestätigen Phagengenom Isolation, Lanes: 1. Lambda Integrase (750 bp), 2. Lambda-Repressor (356 bp) 3. Lambda SIE (Superinfektion Ausschluss) (456 bp) 4. TrackIt 1 kb zuzüglich Leiter (Invitrogen). <img alt="Fig. 4" src="/files/ftp_upload/3899/3899fig4.jpg" /> Abbildung 4. Lambda Genoms Attribute und Berichterstattung. A) GC Grundstück, B) Genome Karte von Lambda (adaptiert aus <a href="http://img.jgi.doe.gov" target="_blank">http://img.jgi.doe.gov</a> ) und C) Mapping von SVG liest der Referenz Bakteriophagen Lambda, ist x-Achse Genom Position, yaxis ist% Deckung.

<ol>
	<li>Raghunathan, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+DNA+amplification+from+a+single+bacterium.">Genomic DNA amplification from a single bacterium.</a> Appl. Environ. Microbiol. 71, 3342-337 (2005).</li><li>Lasken, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+genomic+sequencing+using+Multiple+Displacement+Amplification.">Single-cell genomic sequencing using Multiple Displacement Amplification.</a> Curr. Opin. Microbiol. 10, 510-516 (2007).</li><li>Ishoey, T., Woyke, T., Stepanauskas, R., Novotny, M., Lasken, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+sequencing+of+single+microbial+cells+from+environmental+samples.">Genomic sequencing of single microbial cells from environmental samples.</a> Curr. Opin. Microbiol. 11, 198-204 (2008).</li><li>Edwards, R. A., Rohwer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+metagenomics.">Viral metagenomics.</a> Nature Reviews Microbiology. 3, 504-510 (2005).</li><li>Rohwer, F., Prangishvili, D., Lindell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+viruses+in+the+environment.">Roles of viruses in the environment.</a> Environ. Microbiol. 11, 2771-2774 (2009).</li><li>Williamson, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Sorcerer+II+Global+Ocean+Sampling+Expedition:+metagenomic+characterization+of+viruses+within+aquatic+microbial+samples.">The Sorcerer II Global Ocean Sampling Expedition: metagenomic characterization of viruses within aquatic microbial samples.</a> PLoS ONE. 3, e1456 (2008).</li><li>John, S. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+efficient+method+for+concentration+of+ocean+viruses+by+chemical+flocculation.">A simple and efficient method for concentration of ocean viruses by chemical flocculation.</a> Environ. Microbiol. Rep. 3, 195-202 (2011).</li><li>Noble, R., Fuhrman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+SYBR+Green+I+for+rapid+epifluorescence+counts+of+marine+viruses+and+bacteria.">Use of SYBR Green I for rapid epifluorescence counts of marine viruses and bacteria.</a> Aquat Microb Ecol. 14, 113-118 (1998).</li><li>Hara, S., Terauchi, K., Koike, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abundance+of+viruses+in+marine+waters:+assessment+by+epifluorescence+and+transmission+electron+microscopy.">Abundance of viruses in marine waters: assessment by epifluorescence and transmission electron microscopy.</a> Appl. Environ. Microbiol. 57, 2731-2734 (1991).</li><li>Hennes, K. P., Suttle, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+counts+of+viruses+in+natural+waters+and+laboratory+cultures+by+epifluorescence+microscopy.">Direct counts of viruses in natural waters and laboratory cultures by epifluorescence microscopy.</a> Limnol. Oceanogr. 40, 1050-1055 (1995).</li><li>Marie, D., Brussaard, C. P. D., Thyrhaug, R., Bratbak, G., Vaulot, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enumeration+of+marine+viruses+in+culture+and+natural+samples+by+flow+cytometry.">Enumeration of marine viruses in culture and natural samples by flow cytometry.</a> Appl. Environ. Microbiol. 65, (1999).</li><li>Brussaard, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enumeration+of+bacteriophages+using+flow+cytometry.">Enumeration of bacteriophages using flow cytometry.</a> Methods Mol. Biol. 501, 97-111 (2009).</li><li>Allen, L. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Virus+Genomics:+A+New+Tool+for+Virus+Discovery.">Single Virus Genomics: A New Tool for Virus Discovery.</a> Plos One. 6, (2011).</li><li>Hutchison, C. A., Smith, H. O., Pfannkoch, C., Venter, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+cloning+using+phi+29+DNA+polymerase.">Cell-free cloning using phi 29 DNA polymerase.</a> Proceedings of the National Academy of Sciences of the United States of America. 102, 17332-17336 (2005).</li></ol>

Keine Interessenskonflikte erklärt.

Eine Vielzahl wichtiger Faktoren müssen berücksichtigt werden, wenn die Anwendung SVG Ansätze. Genotypisierung, so wurde während des Proof-of-Concept-Experiment 13 durchgeführt wird, ist keine gültige Option für die Umwelt oder die unbekannten Isolate als konservierte Primer nicht verfügbar sind, in allen viralen Gruppen. Auch ist Hintergrund DNA-Synthese oder unspezifische Amplifikation häufig während der Amplifikation mit dem MDA-Reaktion 14 gemeldet. Die unspezifische Amplifikation kon...

<table border="1">
	<tbody>
		<tr>
			<td>Material Name</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>1X TE buffer</td>
			<td>Invitrogen</td>
			<td>12090-015</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Buffer-saturated Phenol</td>
			<td>Invitrogen</td>
			<td>15513-039</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>GenomiPhi HY kit</td>
			<td>GE Healthcare</td>
			<td>25-6600-22</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glycoblue</td>
			<td>Invitrogen</td>
			<td>AM9516</td>
			<td>15 mg/ml</td>
		</tr>
		<tr>
			<td>LMP agarose</td>
			<td>Invitrogen</td>
			<td>16520100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PTFE microscope slide</td>
			<td>Electron Microscopy Sciences</td>
			<td>63430-04</td>
			<td>24 well, 4 mm Diameter</td>
		</tr>
		<tr>
			<td>SybrGreen</td>
			<td>Invitrogen</td>
			<td>S7585</td>
			<td>10,000X</td>
		</tr>
		<tr>
			<td>&beta;-agarase</td>
			<td>New England Biolabs</td>
			<td>M0392S</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

isolation and genome analysis of single virions using 'single virus genomics'

Amplifikation des gesamten Genoms und Aneinanderreihung einzelner mikrobiellen Zellen ermöglicht Genoms ohne die Notwendigkeit des Anbaus 1-3. Viren, die allgegenwärtig und die meisten zahlreiche Unternehmen auf unserem Planeten 4 und wichtig in allen Umgebungen 5 sind, müssen noch über ähnliche Ansätze aufgedeckt werden. Hier beschreiben wir einen Ansatz für die Isolierung und Charakterisierung der Genome einzelner Virionen genannt &#39;Single Virus Genomics &quot;(SVG). SVG verwendet Durchflusszytometrie einzelnen Viren, Amplifikation des gesamten Genoms zu isolieren, um hochmolekulare genomische DNA (gDNA), die in nachfolgenden Reaktionen Sequenzierung verwendet werden können erhalten.

Watch this Scientific Journal Video about Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics' at JoVE.com

Isolation and Genome Analysis of Single Virions using &#039;Single Virus Genomics&#039;

Einzel-Virus Genomics (SVG) ist eine Methode zur Isolierung und verstärken die Genome einzelner virons. Virussuspensionen einer gemischten Anordnung sortiert werden mittels Durchflusszytometrie auf einen Objektträger mit diskreten Vertiefungen, Agarose, wodurch die Erfassung des Virions und Verringerung Genom Scherung während der Weiterverarbeitung. Amplifikation des gesamten Genoms erfolgt über mehrere Displacement Amplification (MDA), was genomische Material, das für die Sequenzierung geeignete ist.

Isolation und Genomanalyse Einzel Virionen mit &#39;Single Virus Genomics&#39;

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

isolation-genome-analysis-single-virions-using-single-virus

Research

JoVE Journal

Immunology and Infection

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

10.8K Aufrufe.  The J. Craig Venter Institute. Einzel-Virus Genomics (SVG) ist eine Methode zur Isolierung und verstärken die Genome einzelner virons. Virussuspensionen einer gemischten Anordnung sortiert werden mittels Durchflusszytometrie auf einen Objektträger mit diskreten Vertiefungen, Agarose, wodurch die Erfassung des Virions und Verringerung Genom Scherung während der Weiterverarbeitung. Amplifikation des gesamten Genoms erfolgt über mehrere Displacement Amplification (MDA), was genomische Material, das f?...

Serie: Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in bioterrorism is also a concern. A better understanding of the cellular factors that impact infection would facilitate the development of much-needed therapeutics. Recent advances in RNA interference (RNAi) technology coupled with complete genome sequencing of several organisms has led to the optimization of genome-wide, cell-based loss-of-function screens. Drosophila cells are particularly amenable to genome-scale screens because of the ease and efficiency of RNAi in this system 1. Importantly, a wide variety of viruses can infect Drosophila cells, including a number of mammalian viruses of medical and agricultural importance 2,3,4. Previous RNAi screens in Drosophila have identified host factors that are required for various steps in virus infection including entry, translation and RNA replication 5. Moreover, many of the cellular factors required for viral replication in Drosophila cell culture are also limiting in human cells infected with these viruses 4,6,7,8, 9. Therefore, the identification of host factors co-opted during viral infection presents novel targets for antiviral therapeutics. Here we present a generalized protocol for a high-throughput RNAi screen to identify cellular factors involved in viral infection, using vaccinia virus as an example.

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.

RNAi Screening für Host beteiligten Faktoren in Vaccinia Virus-Infektion mit Drosophila Cells

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in ...

Forschung

Immunologie und Infektion

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes 3. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. 
Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes 4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates 10. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community&#x2019;s genetic signature. 
Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s11. By exploiting the charge interaction between the positively-charged Ca2+ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing 12. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography.

We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.

Trennung von Einzelstrang-DNA, doppelsträngige DNA und RNA aus ökologischer Viral Gemeinschaft mit Hydroxyapatit-Chromatographie

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

Einzel-Zell-Analyse Bacillus subtilis Biofilme mittels Fluoreszenzmikroskopie und Durchflusszytometrie

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome organization are governed by DNA-binding proteins that recognize specific DNA structures or sequences. In vitro experiments have helped to generate detailed models for the function of many types of DNA-binding proteins, yet, the exact mechanisms of these processes and their organization in the complex environment of the living cell remain far less understood. We recently introduced a method for quantifying DNA-repair activities in live Escherichia coli cells using Photoactivated Localization Microscopy (PALM) combined with single-molecule tracking. Our general approach identifies individual DNA-binding events by the change in the mobility of a single protein upon association with the chromosome. The fraction of bound molecules provides a direct quantitative measure for the protein activity and abundance of substrates or binding sites at the single-cell level. Here, we describe the concept of the method and demonstrate sample preparation, data acquisition, and data analysis procedures.

Photoactivated localization microscopy (PALM) combined with single-molecule tracking allows direct observation and quantification of protein-DNA interactions in live Escherichia coli cells.

Visualisierung von Protein-DNA-Wechselwirkungen in der lebenden Bakterienzellen Verwendung Photoactivated Einzelmolekül-Tracking

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome ...

Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and epidermis home a variety of immune cells in both homeostatic and inflammatory conditions. Tools to obtain skin cell release for cytofluorimetric analyses are, therefore, very useful in order to study the complex network of immune cells residing in the skin and their response to microbial stimuli. Here, we describe an efficient methodology for the digestion of mouse skin to rapidly and efficiently obtain single-cell suspensions. This protocol allows maintenance of maximum cell viability without compromising surface antigen expression. We also describe how to take and digest skin samples from different anatomical locations, such as the ear, trunk, tail, and footpad. The obtained suspensions are then stained and analyzed by flow cytometry to discriminate between different leukocyte populations.

The skin is home to a complex immune cell network. We describe an efficient methodology for the digestion of mouse skin, from different parts of the animal's body, in order to obtain a single-cell suspension and analyze the different leukocyte populations resident in the skin by flow cytometry.

Herstellung von Einzelzellsuspensionen für Cytofluorimetric Analyse von verschiedenen Mäusehaut Regionen

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophilen-Isolierung und Analyse zur Bestimmung ihrer Rolle in der Lymphom-Zellempfindlichkeit therapeutische Mittel

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and multiplexing ability. Using fluorescent nano-diamond fiducial markers, we describe the drift correction and alignment procedures required to obtain high precision in single molecule localization microscopy. In addition, a new multiplexing strategy, madSTORM, is described in which multiple molecules are targeted in the same cell using sequential binding and elution of fluorescent antibodies. madSTORM is demonstrated on an activated T cell to visualize the locations of different components within a membrane-bound, multi-protein structure called the T cell receptor microcluster. In addition, application of madSTORM as a general tool for visualization of multi-protein structures is discussed.

We demonstrate a method to image multiple molecules within heterogeneous nano-structures at single molecule accuracy using sequential binding and elution of fluorescently labeled antibodies.

Hochmultiplexed, Super-Auflösung Bildgebung von T-Zellen mit madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and ...

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be difficult. To overcome this challenge, a methodology combining indexed flow cytometry and high-throughput multiplexed quantitative polymerase chain reaction (qPCR) was developed. The objective was to identify and characterize simian immunodeficiency virus (SIV)-infected cells present within rhesus macaques. Through quantitation of surface protein by fluorescence-activated cell sorting (FACS) and mRNA by qPCR, virus-infected cells are identified by viral gene expression, which is combined with host gene and protein measurements to create a multidimensional profile. We term the approach, targeted Single-Cell Proteo-transcriptional Evaluation, or tSCEPTRE. To perform the method, viable cells are stained with fluorescent antibodies specific for surface markers used for FACS isolation of a cell subset and/or downstream phenotypic analysis. Single cells are sorted followed by immediate lysis, multiplex reverse transcription (RT), PCR pre-amplification, and high throughput qPCR of up to 96 transcripts. FACS measurements are recorded at the time of sorting and subsequently linked to the gene expression data by well position to create a combined protein and transcriptional profile. To study SIV-infected cells directly ex vivo, cells were identified by qPCR detection of multiple viral RNA species. The combination of viral transcripts and the quantity of each provide a framework for classifying cells into distinct stages of the viral life cycle (e.g., productive versus non-productive). Moreover, tSCEPTRE of SIV+ cells were compared to uninfected cells isolated from the same specimen to assess differentially expressed host genes and proteins. The analysis revealed previously unappreciated viral RNA expression heterogeneity among infected cells as well as in vivo SIV-mediated post-transcriptional gene regulation with single-cell resolution. The tSCEPTRE method is relevant for the analysis of any cell population amenable to identification by expression of surface protein marker(s), host or pathogen gene(s), or combinations thereof.

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Described is a methodology to quantitate the expression of 96 genes and 18 surface proteins by single cells ex vivo, allowing for the identification of differentially expressed genes and proteins in virus-infected cells relative to uninfected cells. We apply the approach to study SIV-infected CD4+ T cells isolated from rhesus macaques.

Einzellige Quantifizierung der mRNA und Oberfläche Protein-Expression in Simian Immunodeficiency Virus-infizierten CD4+ T Zellen isoliert von Rhesus-Makaken

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be ...

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies in an attempt to understand the precise mechanisms that lead to the breakdown of self-tolerance and subsequent autoimmunity. Approaches thus far have been based on the study of one specific aspect of the immune system (a single or few cell types or cytokines), and do not offer a global assessment of complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected. A comprehensive single-cell approach to evaluate cytokine production in multiple immune cell subsets, within the context of &#34;intrinsic&#34; patient-specific plasma circulating factors, is described here. This approach enables monitoring of the patient-specific immune phenotype (surface markers) and function (cytokines), either in its native &#34;intrinsic pathogenic&#34; disease state, or in the presence of therapeutic agents (in vivo or ex vivo).

Here we describe a single-cell proteomic approach to evaluate immune phenotypic and functional (intracellular cytokine induction) alterations in peripheral whole blood samples, analyzed via mass cytometry.

Einzellige Analyse der Immunophenotype und Produktion von Zytokinen im peripheren Blut über Masse Zytometrie

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected in the urinary tract. The protocol can be broadly divided into three sections: the infection, bladder epithelial cell harvesting, and mouth micropipetting to isolate individual infected epithelial cells. The isolated epithelial cell contains viable bacterial cells and is nearly free of contaminating extracellular bacteria, making it ideal for downstream single-cell analysis. The time taken from the start of infection to obtaining a single intracellular bacterial community is about 8 h. This protocol is inexpensive to deploy and uses widely available materials, and we anticipate that it can also be utilized in other infection models to isolate single infected cells from cell mixtures even if those infected cells are rare. However, due to a potential risk in mouth micropipetting, this procedure is not recommended for highly infectious agents.

This protocol describes a simple method of isolating single, infected-bladder epithelial cells from a murine model of urinary tract infection.

Isolierung der einzelnen intrazelluläre bakterielle Gemeinschaften erzeugt aus einem Mausmodell der urinausscheidende Fläche Infektion nachgelagerten Einzeller Analysezwecken

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected ...