Damos las gracias a Sergei Demin y Karamysheva Tatyana por su ayuda en la preparación de los cromosomas y FISH en Anopheles. También agradecemos a David Severson por darnos Aedes y Culex ADN genómico clones BAC y Wade Melissa para editar el texto. Este trabajo fue apoyado por dos subvenciones de los Institutos Nacionales de Salud: 1R21 AI88035-01 a Maria V. Sharakhova y 1R21 AI094289-01 a Igor V. Sharakhov.

<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>

No hay conflictos de interés declarado.

No fluorescentes de hibridación in situ en cromosomas mitóticos de mosquitos se realizó por primera vez en 1990 por A. Kumar y K. Rai 17. En ese estudio, 18S y los genes 28S ribosomal DNA clonado, juntos en un plásmido, se colocaron a los cromosomas de las 20 especies de mosquitos. La sonda de ADN se marcó radiactivamente y se hibridaron a los cromosomas de los ganglios de cerebro. Entre los tres géneros de mosquitos, una técnica de FISH se ha desarrollado sólo para los cromosomas mitóticos a partir de la línea celular de Ae. aegypti 10,18,19 y nunca se ha realizado en los cromosomas mitóticos de mosquitos vivos. Recientemente, hemos desarrollado una técnica sencilla y robusta para obtener alta calidad preparaciones de cromosomas de las IDs de 4 º estadio las larvas 9. Este método permite un elevado número de cromosomas que se obtenga en una diapositiva y puede ser utilizado universalmente para todas las especies de mosquitos. La necesidad de utilizar sta no sólo las larvas, pupa o adultoGES de mosquitos, para la preparación de diapositivas es probablemente la única limitación del método. El método FISH estándar 12 se ha optimizado para el uso de genómica clon BAC y rDNA IGS como sondas para los cromosomas mitóticos de Aedes, Culex, y Anopheles. Además de estas aplicaciones específicas, los protocolos de FISH descritos aquí también se puede utilizar para otros fines. El protocolo FISH avanzada, que utiliza C 0 fracciones de ADN T para bloquear la hibridación no específica, también se puede aplicar para la hibridación de clones BAC o cualquier otros fragmentos grandes de ADN en las regiones heterocromáticas de Anopheles. Heterochromatic regiones se enriquecen con elementos transponibles y repite otras, y las sondas de estas regiones producen normalmente sólida formación en los 3 cromosomas. Uso no etiquetados C 0 fracciones de ADN T ayudará a reducir la hibridación no específica de la sonda a los cromosomas. La versión simple del FISH protocol puede ser utilizado para cualquier rDNA o sondas de ADN repetitivo en los cromosomas mitóticos de mosquitos y otros insectos. Además, también se puede aplicar para la hibridación de ADN clon BAC en las especies con bajo contenido de ADN repetitivo en euchromatic regiones tales como Anopheles o Drosophila. El protocolo propuesto aquí le ayudará a obtener altamente terminados basados ​​en el cromosoma genoma asambleas para los mosquitos y puede ser ampliamente utilizado para aplicaciones distintas citogenéticas en otros grupos de insectos.

<table border="1"><tbody><tr><td> Nombre del reactivo / Equipo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> MZ6 Leica microscopio estereoscópico </td><td> Leica </td><td> VA-OM-E194-354 </td><td> Un microscopio estereoscópico se pueden utilizar diferentes </td></tr><tr><td> Olympus CX41 fase microscopio </td><td> Olimpo </td><td> CX41 </td><td> Un microscopio de fase diferente se puede utilizar </td></tr><tr><td> Olympus BX61 microscopio de fluorescencia </td><td> Olimpo </td><td> BX61 </td><td> Un microscopio fluorescente diferente se puede utilizar </td></tr><tr><td> Slide ThermoBrite desnaturalización / hibridación Sistema </td><td> Abbott Molecular </td><td> 30-144110 </td><td> Sirve como un bloque de calentamiento y una cámara húmeda </td></tr><tr><td> Agujas de disección </td><td> ScienceTools Artes </td><td> 10130-10</td><td></td></tr><tr><td> Los porta-agujas </td><td> Herramientas Artes Ciencias </td><td> 26018-17 </td><td></td></tr><tr><td> Tijeras de disección </td><td> Herramientas Artes Ciencias </td><td> 15000-03 </td><td></td></tr><tr><td> 75x25 doble micro esmerilado se desliza </td><td> Corning </td><td> 2949-75x25 </td><td></td></tr><tr><td> 22x22 mm cubreobjetos microscopio </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> Adhesivo de goma </td><td> Fisher Scientific </td><td> 50-949-105 </td><td></td></tr><tr><td> Ácido acético </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> El ácido propiónico </td><td> Sigma-Aldrich </td><td> 402907 </td><td></td></tr><tr><td> Ácido clorhídrico </td><td> Fisher Scientific </td><td> A144-500 </td><td></td></tr><tr><td> Sodio dihidrato de citrato </td><td> Fisher Scientific </td><td> S279-500 </td><td></td></tr><tr><td> Trihidrato de acetato de sodio </td><td> Fisher Scientific </td><td> BP334-500 </td><td></td></tr><tr><td> El cloruro de potasio </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 (formalina tamponada neutra) </td><td> Sigma-Aldrich </td><td> HT501128 </td><td></td></tr><tr><td> 99% de formamida </td><td> Fisher Scientific </td><td> BP227500 </td><td></td></tr><tr><td> El sulfato de dextrano sódico sal </td><td> Sigma-Aldrich </td><td> D8906 </td><td></td></tr><tr><td> 20x tampón SSC </td><td> Invitrogen </td><td> AM9765 </td><td></td></tr><tr><td> 1 mM de yoduro YOYO-1 (491/509) solución </td><td> Invitrogen </td><td> Y3601 </td><td></td></tr><tr><td> Antifade Prolongar reactivo Oro </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> ADN Polimerasa I </td><td> Fermentas </td><td> EP0041 </td><td></td></tr><tr><td> DNasa I </td><td> Fermentas </td><td> EN0521 </td><td></td></tr><tr><td> Nucleasa S1 </td><td> Fermentas </td><td> EN0321 </td><td></td></tr><tr><td> Taq ADN polimerasa </td><td> Invitrogen </td><td> 18038-042 </td><td></td></tr><tr><td> RNasa </td><td> Sigma-Aldrich </td><td> 9001-99-4 </td><td></td></tr><tr><td> Pepsina </td><td> USB </td><td> 9001-75-6 </td><td></td></tr><tr><td> ADN de esperma de salmón </td><td> Sigma-Aldrich </td><td> D7656 </td><td></td></tr><tr><td> Nonidet-P40 (NP40) </td><td> EE.UU. Biológica </td><td> NC9375914 </td><td></td></tr><tr><td> Qiagen Blood y Cell Culture Maxikit </td><td> Qiagen </td><td> 13362 </td><td></td></tr><tr><td> Qiagen Kit construir grandes </td><td> Qiagen </td><td> 12462 </td&gt; <td></td></tr></tbody></table>

fluorescent in situ hybridization on mitotic chromosomes of mosquitoes

La hibridación fluorescente in situ (FISH) es una técnica utilizada habitualmente por muchos laboratorios para determinar la posición cromosómica de sondas de ADN y ARN. Una aplicación importante de este método es el desarrollo de mapas de alta calidad físicos útiles para mejorar los conjuntos de genoma de varios organismos. El patrón natural de bandas de los cromosomas mitóticos politénicos y proporciona orientación para la ordenación y orientación precisas de los supercontigs genómicos. Entre los tres géneros de mosquitos Anopheles, a saber, Aedes, Culex y, una bien establecida cromosoma técnica basada en la cartografía se ha desarrollado sólo para Anopheles, cuyos miembros poseen cromosomas legibles politénicos 1. Como resultado de los esfuerzos de mapeo del genoma, el 88% de la Un. gambiae genoma se ha colocado para cromosoma posiciones precisas 2,3. Dos géneros de mosquitos Aedes y otros, Culex, han mal polytenized b cromosomasomo de la sobrerrepresentación significativa de elementos transponibles en sus genomas 4, 5, 6. Sólo 31 y 9% de los supercontings genómicas se han asignado sin orden o la orientación de los cromosomas de Ae. aegypti 7 y Cx. quinquefasciatus 8, respectivamente. Preparación mitótica cromosoma para estas dos especies previamente había sido limitada a los ganglios cerebro y líneas celulares. Sin embargo, los portaobjetos cromosómicos preparados a partir de los ganglios cerebro de los mosquitos por lo general contienen un bajo número de placas de metafase 9. Además, aunque una técnica de FISH se ha desarrollado para cromosomas mitóticos de una línea celular de Ae. aegypti 10, la acumulación de múltiples reordenamientos cromosómicos en los cromosomas de líneas celulares 11 los hace inútiles para la cartografía del genoma. Aquí se describe una técnica sencilla y robusta para obtener alta calidad preparaciones de cromosomas mitóticos de los discos imaginales (ID) de 4 º estadio las larvas que cuna se utiliza para todos los tres géneros de mosquitos. Un protocolo FISH estándar 12 está optimizado para el uso de clones de BAC de ADN genómico como una sonda en los cromosomas mitóticos de Ae. aegypti y Cx. quinquefasciatus, y para la utilización de un espaciador intergénico (IGS) región de ADN ribosómico (ADNr) como una sonda en una. cromosomas gambiae. Además de la cartografía física, la técnica desarrollada se puede aplicar a la citogenética de la población y la taxonomía cromosoma / sistemática de los mosquitos y otros grupos de insectos.

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

Entre los tres géneros de mosquitos, es decir,<em&gt; Anopheles,</em&gt;<em&gt; Aedes</em&gt; Y<em&gt; Culex</em&gt;, Técnicas de mapeo físico del genoma se crearon sólo para<em&gt; Anopheles</em&gt;, Cuyos miembros poseen cromosomas politénicos legibles. Para los géneros de<em&gt; Aedes</em&gt; Y<em&gt; Culex</em&gt; Sin embargo, la cartografía citogenético sigue siendo un reto debido a la mala calidad de los cromosomas politénicos. Aquí presentamos un protocolo universal para la obtención de preparados de alta calidad de los cromosomas mitóticos y un protocolo FISH optimizado para los tres géneros de mosquitos.

Fluorescente<em&gt; In situ</em&gt; La hibridación en los cromosomas mitóticos de mosquitos

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

25.5K vistas.  Virginia Tech. Entre los tres géneros de mosquitos, es decir, Anopheles, Aedes Y Culex, Técnicas de mapeo físico del genoma se crearon sólo para Anopheles, Cuyos miembros poseen cromosomas politénicos legibles. Para los géneros de Aedes Y Culex Sin embargo, la cartografía citogenético sigue siendo un reto debido a la mala calidad de los cromosomas politénicos. Aquí presentamos un protocol...

Video: 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).

Combinación de adhesivo basado en cinta de muestreo y de fluorescencia In situ La hibridación para la detección rápida de Salmonella En los productos frescos

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

Investigación

Inmunología e Infección

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.

Flujo de ácido nucleico cerrado Citometría de fluorescencia In situ</emHibridación> (LNA flujo-FISH): un método para la detección de bacterias pequeñas de ARN

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.

Hibridación<em&gt; In situ</em&gt; De las glándulas salivales, los ovarios y embriones de los mosquitos vectores

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

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).

Pintura de cromosomas 2D y 3D en mosquitos de la malaria

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

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).

Fluorescencia cuantitativa en hibridación situ (FISH) e inmunofluorescencia (IF) de productos genéticos específicos en células infectadas por KSHV

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.

Visualización de Candida albicans en el tracto gastrointestinal murino utilizando hibridación fluorescente in situ

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.

Análisis de competencia vectorial en mosquitos Aedes aegypti con el virus Zika

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.

Propagación del parásito microsporidiano Edhazardia aedis en mosquitos Aedes aegypti

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

Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in cell line&#160;and three-dimensional (3D) cultures of human airway epithelium. The method allows highly specific and sensitive visualization of viral RNA by relying on HCR initiated by probe localization. Split-initiator probes help amplify the signal by fluorescently labeled amplifiers, resulting in negligible background fluorescence in confocal microscopy. Labeling amplifiers with different fluorescent dyes facilitates the simultaneous recognition of various targets. This, in turn, allows the mapping of the infection in tissues to better understand viral pathogenesis and replication at the single-cell level. Coupling this method with immunofluorescence may facilitate better understanding of host-virus interactions, including alternation of the host epigenome and immune response pathways. Owing to sensitive and specific HCR technology, this protocol can also be used as a diagnostic tool. It is also important to remember that the technique may be modified easily to enable detection of any RNA, including non-coding RNAs and RNA viruses that may emerge in the future.

Here, we describe a simple method that combines RNA fluorescence in situ&#160;hybridization (RNA-FISH) with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. This protocol may increase understanding of the molecular characteristics of SARS-CoV-2 RNA-host interactions at a single-cell level.

Visualización Del SARS-CoV-2 Usando Inmuno ARN-Fluorescencia Hibridación In Situ

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory ...

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota and the vertebrate gut. The spatial landscape of the gut microbiota is tightly controlled by physical features - intestinal mucus, crypts, and folds - and is affected by host-controlled properties such as pH, oxygen availability, and immune factors. These properties limit the ability of commensal microbes and pathogens alike to colonize the gut stably. At the micron-scale, microbial organization determines the close-range interactions between different microbes as well as the interactions between microbes and their host. These interactions then affect large-scale organ function and host health.
This protocol enables the visualization of the gut microbiota spatial organization from distances between cells to organ-wide scales. The method is based on fixing gut tissues while preserving intestinal structure and mucus properties. The fixed samples are then embedded, sectioned, and stained to highlight specific bacterial species through fluorescence in situ hybridization (FISH). Host features, such as mucus and host cell components, are labeled with fluorescently labeled lectins. Finally, the stained sections are imaged using a confocal microscope utilizing tile-scan imaging at high magnification to bridge the micron to centimeter length scales. This type of imaging can be applied to intestinal sections from animal models and biopsies from human tissues to determine the biogeography of the microbiota in the gut in health and disease.

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

This streamlined protocol details a workflow to detect and image bacteria in complex tissue samples, from fixing the tissue to staining microbes with fluorescent in situ hybridization.

Visualización de las interacciones entre la microbiota intestinal y el huésped a través de la hibridación fluorescente in situ , la tinción de lectina y la obtención de imágenes

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota ...