Wir danken Sergei Demin und Tatjana Karamysheva für ihre Hilfe bei Chromosom Vorbereitung und FISH auf Anopheles. Wir danken auch David Severson für die Bereitstellung Aedes und Culex genomische DNA BAC-Klone und Melissa Wade für die Bearbeitung des Textes. 1R21 AI88035-01 bis Maria V. Sharakhova und 1R21 AI094289-01 bis Igor V. Sharakhov: Diese Arbeit wurde durch zwei Zuschüsse aus dem National Institutes of Health unterstützt.

<ol>
	<li>Sharakhov, I. V., Sharakhova, M. V., Verrity, J. F., Abbington, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chromosome Mapping Research Developments. , (2008).</li><li>Holt, R. 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=The+genome+sequence+of+the+malaria+mosquito+Anopheles+gambiae.">The genome sequence of the malaria mosquito Anopheles gambiae.</a> Science. 298, 129-149 (2002).</li><li>Sharakhova, M. V., 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=Update+of+the+Anopheles+gambiae+PEST+genome+assembly.">Update of the Anopheles gambiae PEST genome assembly.</a> Genome biology. 8, R5 (2007).</li><li>Campos, J., Andrade, C. F., Recco-Pimentel, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technique+for+preparing+polytene+chromosomes+from+Aedes+aegypti+(Diptera,+Culicinae).">A technique for preparing polytene chromosomes from Aedes aegypti (Diptera, Culicinae).</a> Memorias do Instituto Oswaldo Cruz. 98, 387-390 (2003).</li><li>Campos, J., Andrade, C. F., Recco-Pimentel, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malpighian+tubule+polytene+chromosomes+of+Culex+quinquefasciatus+(Diptera,+Culicinae).">Malpighian tubule polytene chromosomes of Culex quinquefasciatus (Diptera, Culicinae).</a> Memorias do Instituto Oswaldo Cruz. 98, 383-386 (2003).</li><li>McAbee, R. D., Christiansen, J. A., Cornel, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+detailed+larval+salivary+gland+polytene+chromosome+photomap+for+Culex+quinquefasciatus+(Diptera:+Culicidae)+from+Johannesburg,+South+Africa.">A detailed larval salivary gland polytene chromosome photomap for Culex quinquefasciatus (Diptera: Culicidae) from Johannesburg, South Africa.</a> J. Med. Entomol. 44, 229-237 (2007).</li><li>Nene, V., 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=Genome+sequence+of+Aedes+aegypti,+a+major+arbovirus+vector.">Genome sequence of Aedes aegypti, a major arbovirus vector.</a> Science. 316, 1718-1723 (2007).</li><li>Arensburger, P., 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=Sequencing+of+Culex+quinquefasciatus+establishes+a+platform+for+mosquito+comparative+genomics.">Sequencing of Culex quinquefasciatus establishes a platform for mosquito comparative genomics.</a> Science. 330, 86-88 (2010).</li><li>Sharakhova, M. V., 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=Imaginal+discs--a+new+source+of+chromosomes+for+genome+mapping+of+the+yellow+fever+mosquito+Aedes+aegypti.">Imaginal discs--a new source of chromosomes for genome mapping of the yellow fever mosquito Aedes aegypti.</a> PLoS neglected tropical diseases. 5, e1335 (2011).</li><li>Brown, S. E., 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=Toward+a+physical+map+of+Aedes+aegypti.">Toward a physical map of Aedes aegypti.</a> Insect Mol. Biol. 4, 161-167 (1995).</li><li>Steiniger, G. E., Mukherjee, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+chromosome+banding:+technique+for+G-+and+Q-banding+pattern+in+the+mosquito+Aedes+albopictus.">Insect chromosome banding: technique for G- and Q-banding pattern in the mosquito Aedes albopictus.</a> Can. J. Genet. Cytol. 17, 241-244 (1975).</li><li>Garimberti, E., Tosi, S., Bridger, J. M., Volpi, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fluorescence in situ hybridization (FISH). , (2010).</li><li>Scott, J. A., Brogdon, W. G., Collins, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+single+specimens+of+the+Anopheles+gambiae+complex+by+the+polymerase+chain+reaction.">Identification of single specimens of the Anopheles gambiae complex by the polymerase chain reaction.</a> The American journal of tropical medicine and hygiene. 49, 520-529 (1993).</li><li>Trifonov, V. A., Vorobyeva, N. N., Rens, W., Leiehr, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fluorescence in situ hybridization (FISH). , (2009).</li><li>Collins, F. H., 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+ribosomal+RNA+gene+probe+differentiates+member+species+of+the+Anopheles+gambiae+complex.">A ribosomal RNA gene probe differentiates member species of the Anopheles gambiae complex.</a> The American journal of tropical medicine and hygiene. 37, 37-41 (1987).</li><li>Kumar, A., Rai, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+localization+and+copy+number+of+18S+28S+ribosomal+RNA+genes+in+evolutionary+diverse+mosquitoes+(Diptera,+Culicidae).">Chromosomal localization and copy number of 18S+28S ribosomal RNA genes in evolutionary diverse mosquitoes (Diptera, Culicidae).</a> Hereditas. 113, 277-289 (1990).</li><li>Brown, S. E., Knudson, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FISH+landmarks+for+Aedes+aegypti+chromosomes.">FISH landmarks for Aedes aegypti chromosomes.</a> Insect Mol. Biol. 6, 197-202 (1997).</li><li>Brown, S. E., Severson, D. W., Smith, L. A., Knudson, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+the+Aedes+aegypti+mosquito+genetic+linkage+and+physical+maps.">Integration of the Aedes aegypti mosquito genetic linkage and physical maps.</a> Genetics. 157, 1299-1305 (2001).</li></ol>

Keine Interessenskonflikte erklärt.

Nichtfluoreszierenden in situ Hybridisierung auf mitotischen Chromosomen von Moskitos wurde erstmals 1990 von A. Kumar und K. Rai 17 durchgeführt. In dieser Studie ribosomalen 18S und 28S-DNA-Gene, zusammen in einem Plasmid kloniert wird, wurden auf den Chromosomen 20 Stechmückenarten platziert. Die DNA-Sonde wurde radioaktiv markiert und hybridisiert an die Chromosomen aus Gehirn Ganglien. Zwischen drei Moskito Gattungen wurde eine FISH-Technik nur für mitotischen Chromosomen von der Zelllinie von Ae entwickelt. aegypti 10,18,19 und hat noch nie auf mitotische Chromosomen von Live-Mücken durchgeführt. Kürzlich entwickelten wir eine einfache, robuste Technik für den Erhalt hochwertiger Chromosom Zubereitungen aus IDs von 4 th Larven 9. Diese Methode ermöglicht eine hohe Anzahl von Chromosomen in eine Folie erhalten werden und ist universell für alle Arten von Stechmücken verwendet werden. Die Notwendigkeit der Verwendung von nur Larven, nicht Puppen oder Erwachsener stages von Mücken, zum Präparat ist wahrscheinlich die einzige Einschränkung des Verfahrens. Die Standard-FISH-Methode 12 wurde für die Verwendung von genomischen BAC-Klon und IGS rDNA als Sonden für den mitotischen Chromosomen Aedes, Culex und Anopheles optimiert. Zusätzlich zu diesen spezifischen Anwendungen können die FISH-Protokolle hier beschriebenen auch für andere Zwecke verwendet werden. Die fortgeschrittene FISH-Protokoll, das C 0 t DNA Fraktionen zur Blockierung unspezifischer Hybridisierung verwendet, können auch für die Hybridisierung von BAC-Klonen oder anderen großen DNA-Fragmente in heterochromatischen Regionen Anopheles aufgebracht werden. Heterochrome Regionen mit Transposons und andere Wiederholungen angereichert und Sonden aus diesen Regionen produzieren normalerweise starken Hintergrund auf den Chromosomen 3. Verwendung unmarkiert C 0 t DNA Fraktionen werden dazu beitragen, unspezifische Hybridisierung der Sonde an die Chromosomen reduzieren. Die einfache Version des FISH protocol kann für jede rDNA oder sich wiederholende DNA-Sonden auf mitotischen Chromosomen von Mücken und anderen Insekten verwendet werden. Darüber hinaus kann es auch für die Hybridisierung von DNA-BAC-Klons in Spezies mit geringem repetitiven DNA-Gehalt in euchromatischen Regionen wie Anopheles oder Drosophila angewendet werden. Die hier vorgeschlagene Protokoll wird dazu beitragen, hoch fertigen Chromosom Genom-basierten Baugruppen für Mücken zu erhalten, und können allgemein für verschiedene zytogenetische Anwendungen in anderen Gruppen von Insekten verwendet werden.

<table border="1"><tbody><tr><td> Name des Reagenz / Equipment </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> MZ6 Leica Stereomikroskop </td><td> Leica </td><td> VA-OM-E194-354 </td><td> Eine andere Stereomikroskop benutzt werden </td></tr><tr><td> Olympus CX41 Phasenkontrastmikroskop </td><td> Olymp </td><td> CX41 </td><td> Eine andere Phase Mikroskop kann verwendet werden </td></tr><tr><td> Olympus BX61 Fluoreszenzmikroskop </td><td> Olymp </td><td> BX61 </td><td> Eine andere Fluoreszenzmikroskop benutzt werden </td></tr><tr><td> ThermoBrite Slide Denaturierung / Hybridization System </td><td> Abbott Molecular </td><td> 30-144110 </td><td> Dient als Heizblock und einer feuchten Kammer </td></tr><tr><td> Präpariernadeln </td><td> Feine ScienceTools </td><td> 10130-10</td><td></td></tr><tr><td> Nadelhalter </td><td> Fine Science Tools </td><td> 26018-17 </td><td></td></tr><tr><td> Präparierschere </td><td> Fine Science Tools </td><td> 15000-03 </td><td></td></tr><tr><td> 75x25 Doppel frosted micro gleitet </td><td> Corning </td><td> 2949-75x25 </td><td></td></tr><tr><td> 22x22 mm Mikroskop Deckgläser </td><td> Fisher Scientific </td><td> 12-544-10 </td><td></td></tr><tr><td> Parafilm </td><td> Fisher Scientific </td><td> 13-374-10 </td><td></td></tr><tr><td> Rubber Cement </td><td> Fisher Scientific </td><td> 50-949-105 </td><td></td></tr><tr><td> Essigsäure </td><td> Fisher Scientific </td><td> A491-212 </td><td></td></tr><tr><td> Alcohol 200 Proof </td><td> Decon Laboratories </td><td> 2701 </td><td></td></tr><tr><td> Propionsäure </td><td> Sigma-ALDRIch </td><td> 402907 </td><td></td></tr><tr><td> Salzsäure </td><td> Fisher Scientific </td><td> A144-500 </td><td></td></tr><tr><td> Natriumcitratdihydrat </td><td> Fisher Scientific </td><td> S279-500 </td><td></td></tr><tr><td> Natriumacetattrihydrat </td><td> Fisher Scientific </td><td> BP334-500 </td><td></td></tr><tr><td> Kaliumchlorid </td><td> Fisher Scientific </td><td> BP366-500 </td><td></td></tr><tr><td> EDTA </td><td> Fisher Scientific </td><td> S311-500 </td><td></td></tr><tr><td> Tris-Base </td><td> Fisher Scientific </td><td> BP152-1 </td><td></td></tr><tr><td> 10x PBS </td><td> Invitrogen </td><td> P5493 </td><td></td></tr><tr><td> 10% NBF (neutral gepuffertes Formalin) </td><td> Sigma-Aldrich </td><td> HT501128 </td><td></td></tr><tr><td> 99% Formamid </td><td> Fisher Scientific </td><td> BP227500 </td><td></td></tr><tr><td> Dextransulfat-Natriumsalz </td><td> Sigma-Aldrich </td><td> D8906 </td><td></td></tr><tr><td> 20x SSC-Puffer </td><td> Invitrogen </td><td> AM9765 </td><td></td></tr><tr><td> 1 mM YOYO-1-iodid (491/509)-Lösung </td><td> Invitrogen </td><td> Y3601 </td><td></td></tr><tr><td> Antifade verlängern Gold-Reagenz </td><td> Invitrogen </td><td> <a href="http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&amp;productID=P36930" target="_blank">P36930</a> </td><td></td></tr><tr><td> dATP, dCTP, dGTP, dTTP </td><td> Fermentas </td><td> R0141, R0151, R0161, R0171 </td><td></td></tr><tr><td> Cy3-dUTP, Cy5-dUTP </td><td> GE Healthcare </td><td> PA53022, PA55022 </td><td></td></tr><tr><td> BSA </td><td> Sigma-Aldrich </td><td> A3294 </td><td></td></tr><tr><td> DNA-Polymerase I </td><td> Fermentas </td><td> EP0041 </td><td></td></tr><tr><td> DNase I </td><td> Fermentas </td><td> EN0521 </td><td></td></tr><tr><td> S1 Nuclease </td><td> Fermentas </td><td> EN0321 </td><td></td></tr><tr><td> Taq-DNA-Polymerase </td><td> Invitrogen </td><td> 18038-042 </td><td></td></tr><tr><td> RNase </td><td> Sigma-Aldrich </td><td> 9001-99-4 </td><td></td></tr><tr><td> Pepsin </td><td> USB </td><td> 9001-75-6 </td><td></td></tr><tr><td> Lachssperma-DNA </td><td> Sigma-Aldrich </td><td> D7656 </td><td></td></tr><tr><td> Nonidet-P40 (NP40) </td><td> US Biological </td><td> NC9375914 </td><td></td></tr><tr><td> Qiagen Blood and Cell Culture Maxikit </td><td> Qiagen </td><td> 13362 </td><td></td></tr><tr><td> Qiagen Large Construct Kit </td><td> Qiagen </td><td> 12462 </td&gt; <td></td></tr></tbody></table>

fluorescent in situ hybridization on mitotic chromosomes of mosquitoes

Fluoreszenz-in-situ-Hybridisierung (FISH) ist eine Technik, die routinemäßig von vielen Labors verwendet werden, um die chromosomale Position von DNA und RNA-Sonden zu bestimmen. Eine wichtige Anwendung dieser Methode ist die Entwicklung von hochwertigen physischen Karten nützlich für die Verbesserung der Genom-Baugruppen für verschiedene Organismen. Die natürliche Bandenmuster polytene und mitotische Chromosomen enthält Leitlinien für die präzise Anordnung und Ausrichtung der genomischen supercontigs. Unter den drei Gattungen Moskito, nämlich Anopheles, Aedes und Culex, ein gut etabliertes Chromosomen-Mapping-Technik ist nur für Anopheles, deren Mitglieder besitzen lesbaren Polytänchromosomen 1 entwickelt worden. Als Ergebnis der Bemühungen Genomkartierung, 88% der Ein. gambiae Genoms hat eine präzise Chromosom Positionen 2,3 gelegt. Zwei weitere mosquito Gattungen Aedes und Culex, haben schlecht Chromosomen b polytenizedeil der signifikante Überrepräsentation von Transposons in ihrem Genom 4, 5, 6. Nur 31 bzw. 9% der genomischen supercontings haben ohne Ordnung und Orientierung an Chromosomen Ae zugewiesen wurde. aegypti 7 und Cx. quinquefasciatus 8 auf. Mitotischen Chromosomen Vorbereitung für diese beiden Arten zuvor auf Gehirn Ganglien und Zelllinien beschränkt. Allerdings Chromosom Dias aus dem Gehirn Ganglien Mücken vorbereitet enthalten in der Regel geringe Zahl von Metaphase Platten 9. Außerdem, obwohl eine FISH-Technik wurde für mitotischen Chromosomen aus einer Zelllinie von Ae entwickelt. aegypti 10, macht die Ansammlung von mehreren chromosomalen Umlagerungen in Zelllinie Chromosomen 11 sie nutzlos für die Genom-Mapping. Hier beschreiben wir eine einfache, robuste Technik zur Gewinnung hochwertiger mitotischen Chromosomen Präparationen aus Imaginalscheiben (IDs) von 4 th Larvenstadium die ceine für alle drei Gattungen von Moskitos verwendet werden. Ein Standard-FISH-Protokoll 12 ist für die Verwendung von BAC-Klone von genomischer DNA als eine Sonde auf mitotischen Chromosomen Ae optimiert. aegypti und Cx. quinquefasciatus, und zur Verwendung einer intergenischen Spacer (IGS) Region der ribosomalen DNA (rDNA) als Sonde auf einem. gambiae Chromosomen. Neben physikalische Kartierung, kann die entwickelte Technik zur Bevölkerung Zytogenetik und Chromosom Taxonomie / Systematik der Mücken und andere Insekten Gruppen angewandt werden.

Watch this Scientific Journal Video about Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes at JoVE.com

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Unter den drei Gattungen Moskito, nämlich<em&gt; Anopheles,</em&gt;<em&gt; Aedes</em&gt;, Und<em&gt; Culex</em&gt; Wurden physikalische Kartierung des Genoms Techniken nur für etablierte<em&gt; Anopheles</em&gt; Besitzen, deren Mitglieder lesbar Polytänchromosomen. Für den Gattungen<em&gt; Aedes</em&gt; Und<em&gt; Culex</em&gt; Bleibt jedoch zytogenetische Zuordnung wegen der schlechten Qualität der Polytänchromosomen Herausforderung. Hier präsentieren wir ein universelles Protokoll für den Erhalt hochwertiger Präparate von mitotischen Chromosomen und eine optimierte FISH-Protokoll für alle drei Gattungen von Mücken.

Fluoreszierend<em&gt; In situ</em&gt; Hybridisierung auf mitotischen Chromosomen der Moskitos

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. ...

fluorescent-in-situ-hybridization-on-mitotic-chromosomes-of-mosquitoes

Research

JoVE Journal

Immunology and Infection

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

25.6K Aufrufe.  Virginia Tech. Unter den drei Gattungen Moskito, nämlich Anopheles, Aedes, Und Culex Wurden physikalische Kartierung des Genoms Techniken nur für etablierte Anopheles Besitzen, deren Mitglieder lesbar Polytänchromosomen. Für den Gattungen Aedes Und Culex Bleibt jedoch zytogenetische Zuordnung wegen der schlechten Qualität der Polytänchromosomen Herausforderung. Hier präsentieren wir ein univ...

Serie: Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

Kombination von Klebstoff-tape-basierten Sampling und Fluoreszenz In situ Hybridisierung zum schnellen Nachweis von Salmonellen On Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Forschung

Immunologie und Infektion

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 ...

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes. Investigation of transcripts and gene regulators that are expressed in tissues in which the mosquito host and pathogen interact, and in organs involved in reproduction are of great interest for strategies to reduce mosquito-borne disease transmission and disrupt egg development. A number of tools have been employed to study and validate the temporal and tissue-specific regulation of gene expression. Here, we describe protocols that have been developed to obtain spatial information, which enhances our understanding of where specific genes are expressed and their products accumulate. The protocol described has been used to validate expression and determine accumulation patterns of transcripts in tissues related to mosquito-borne pathogen transmission, such as female salivary glands, as well as subcellular compartments of ovaries and embryos, which relate to mosquito reproduction and development. 
 The following procedures represent an optimized methodology that improves the efficiency of various steps in the protocol without loss of target-specific hybridization signals. Guidelines for RNA probe preparation, dissection of soft tissues and the general procedure for fixation and hybridization are described in Part A, while steps specific for the collection, fixation, pre-hybridization and hybridization of mosquito embryos are detailed in Part B.

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Temporal and spatial gene expression analyses have a crucial role in functional genomics. Whole-mount hybridization in situ is useful for determining the localization of transcripts within tissues and subcellular compartments. Here we outline a hybridization in situ protocol with modifications for specific target tissues in mosquitoes.

Hybridisierung<em&gt; In-situ-</em&gt; Der Speicheldrüsen, Ovarien und Embryonen von Vektor-Mücken

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes.  Investigation of transcripts and gene ...

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

Verwendung Fluoreszierende Proteine, zu visualisieren und zu quantifizieren<em&gt; Chlamydia</em&gt; Vakuole Wachstumsdynamik in lebenden Zellen

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

2D and 3D Chromosome Painting in Malaria Mosquitoes

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting chromosomal rearrangements, and studying three-dimensional (3D) organization of chromosomes in the cell nucleus. The advent of laser capture microdissection (LCM) and whole genome amplification (WGA) allows obtaining large quantities of DNA from single cells. The increased sensitivity of WGA kits prompted us to develop chromosome paints and to use them for exploring chromosome organization and evolution in non-model organisms. Here, we present a simple method for isolating and amplifying the euchromatic segments of single polytene chromosome arms from ovarian nurse cells of the African malaria mosquito Anopheles gambiae. This procedure provides an efficient platform for obtaining chromosome paints, while reducing the overall risk of introducing foreign DNA to the sample. The use of WGA allows for several rounds of re-amplification, resulting in high quantities of DNA that can be utilized for multiple experiments, including 2D and 3D FISH. We demonstrated that the developed chromosome paints can be successfully used to establish the correspondence between euchromatic portions of polytene and mitotic chromosome arms in An. gambiae. Overall, the union of LCM and single-chromosome WGA provides an efficient tool for creating significant amounts of target DNA for future cytogenetic and genomic studies.

Chromosome painting is a useful method for studying organization of the cell nucleus and evolution of the karyotype. Here, we demonstrate an approach to isolate and amplify specific regions of interest from single polytene chromosomes that are subsequently used for two- and three-dimensional fluorescent in situ hybridization (FISH).

2D und 3D Chromosom Malerei in Malaria-Mücken

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

Nicht-invasive Probenahme von Schleimhaut-Flüssigkeit für die Quantifizierung von In Vivo oberen Atemwegen immun-Mediator-Level

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Quantitative Fluorescence In Situ Hybridization (FISH) and Immunofluorescence (IF) of Specific Gene Products in KSHV-Infected Cells

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout the cell at specific times can be captured with fluorescence in situ hybridization (FISH) and immunofluorescence (IF). During lytic herpesvirus infection, the virus hijacks the host cell to preferentially express viral genes, causing changes in cell morphology and behavior of biomolecules. Lytic activities are centered in nuclear factories, termed viral replication compartments, which are discernable only with FISH and IF. Here we describe an adaptable protocol of RNA FISH and IF techniques for Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells, both adherent and in suspension. The method includes steps for the development of specific anti-sense oligonucleotides, double RNA FISH, RNA FISH with IF, and quantitative calculations of fluorescence intensities. This protocol has been successfully applied to multiple cell types, uninfected cells, latent cells, lytic cells, time-courses, and cells treated with inhibitors to analyze the spatiotemporal activities of specific RNAs and proteins from both the human host and KSHV.

Quantitative Fluorescence In Situ Hybridization FISH and Immunofluorescence IF of Specific Gene Products in KSHV-Infected Cells

We describe a protocol utilizing fluorescence in situ hybridization (FISH) to visualize multiple herpesviral RNAs within lytically infected human cells, either in suspension or adherent. This protocol includes quantification of fluorescence producing a nucleocytoplasmic ratio and can be extended for simultaneous visualization of host and viral proteins with immunofluorescence (IF).

Quantitative Fluoreszenz In Situ Hybridisierung (FISH) und Immunfluoreszenz (IF) spezifischer Genprodukte in KSHV-infizierten Zellen

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout ...

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

Visualisierung von Candida albicans im Murine Gastrointestinal Tract mit fluoreszierender In-Situ-Hybridisierung

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine the rate of infection, disseminated infection, and potential transmission of the virus in the mosquito population in question. These procedures are widely utilized with various modifications in vector competence evaluations globally. They are important in determining the potential role that a given mosquito (i.e., species, population, individual) may play in the transmission of a given agent.

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The presented protocol can determine the vector competence of Aedes aegypti mosquito populations for a given virus, such as Zika, in a containment setting.

Vektorkompetenzanalysen an Aedes aegypti Mücken mit Zika-Virus

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine ...

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of disease cases each year. E. aedis causes mortality and reduced reproductive fitness in the mosquito vector and has been explored for its potential as a biocontrol agent. The protocol we present for culturing E. aedis is based on its natural infection cycle, which involves both horizontal and vertical transmission at different life stages of the mosquito host. Ae. aegypti mosquitoes are exposed to spores in the larval stage. These infected larvae then mature into adults and transmit the parasite vertically to their offspring. Infected offspring are then used as a source of spores for future horizontal transmission. Culturing E. aedis can be challenging to the uninitiated given the complexities of the parasite&#8217;s life cycle, and this protocol provides detailed guidance and visual aids for clarification.

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

A protocol to culture the microsporidian parasite Edhazardia aedis. The parasite is passaged from one generation of Aedes aegypti mosquitoes to the next via horizontal transfer at the larval stage followed by vertical transmission at the adult stage. Live sporoplasms survive long-term in infected eggs.

Ausbreitung des mikrosporiden Parasiten Edhazardia aedis in Aedes aegypti Mosquitoes

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of ...