Yazarlar sonrasında uyarlanmış ve hibridizasyon için buraya açıklanan protokolü geliştirmek için modifiye edilmiştir Anofel gambiae embriyoların, in situ hibridizasyon için protokollerin tartışma için yumuşak doku ve Yuri Goltsev in situ yöntemler geliştirmek hibridizasyon tavsiye için Marika Walters teşekkür etmek istiyorum Aedes ve Culex embriyoların yerinde. Ayrıca Adam Pare ve David Kosman tarafından verilen yararlı öneriler kabul ediyorsunuz. Biz bilimsel tartışma Osvaldo Marinotti teşekkür ediyor ve protokol metin düzenleme.

<ol>
	<li>Juhn, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+mapping+of+gene+expression+in+the+salivary+glands+of+the+dengue+vector+mosquito,+Aedes+aegypti.">Spatial mapping of gene expression in the salivary glands of the dengue vector mosquito, Aedes aegypti.</a> Parasit. Vectors. 4, 1 (2011).</li><li>Coleman, J., Juhn, J., James, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+midgut+and+salivary+glands+from+Ae.+aegypti+mosquitoes.">Dissection of midgut and salivary glands from Ae. aegypti mosquitoes.</a> J. Vis. Exp. (5), e228 (2007).</li><li>Benedict, M. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+Plasmodium-infected+Mosquitoes:+Salivary+Glands,+Chapter.+5.4.2.">Dissecting Plasmodium-infected Mosquitoes: Salivary Glands, Chapter. 5.4.2.</a> Methods in Anopheles Research. , (2007).</li><li>Sappington, T. W., Brown, M. R., Raikhel, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+and+analysis+of+insect+ovaries,+Chapter.+42.3.">Culture and analysis of insect ovaries, Chapter. 42.3.</a> The Molecular Biology of Insect Disease Vectors: A methods manual. , (1997).</li><li>Goltsev, Y., Hsiong, W., Lanzaro, G., Levine, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+combinations+of+gap+repressors+for+common+stripes+in+Anopheles+and+Drosophila+embryos.">Different combinations of gap repressors for common stripes in Anopheles and Drosophila embryos.</a> Dev. Biol. 275, 435-446 (2004).</li><li>Benedict, M. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anopheles+Embryo+Fixation,+Chapter.+3.7.">Anopheles Embryo Fixation, Chapter. 3.7.</a> Methods in Anopheles Research. Malaria Research and Reference Reagent Resource Center (MR4). , (2007).</li><li>Juhn, J., James, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=oskar+gene+expression+in+the+vector+mosquitoes,+Anopheles+gambiae+and+Aedes+aegypti.">oskar gene expression in the vector mosquitoes, Anopheles gambiae and Aedes aegypti.</a> Insect Mol. Biol. 15, 363-372 (2006).</li><li>Juhn, J., Marinotti, O., Calvo, E., James, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+structure+and+expression+of+nanos+(nos)+and+oskar+(osk)+orthologues+of+the+vector+mosquito,+Culex+quinquefasciatus.">Gene structure and expression of nanos (nos) and oskar (osk) orthologues of the vector mosquito, Culex quinquefasciatus.</a> Insect Mol. Biol. 17, 545-552 (2008).</li><li>Raminani, L. N., Cupp, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+Embryology+of+Aedes+aegypti+(L.)+(Diptera:+Culicidae).">Early Embryology of Aedes aegypti (L.) (Diptera: Culicidae).</a> Int. J. Insect Morphol. &#38; Embryol. 4, 517-528 (1975).</li><li>Raminani, L. N., Cupp, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryology+of+Aedes+aegypti+(L.)+(Diptera:+Culicidae):+Organogenesis.">Embryology of Aedes aegypti (L.) (Diptera: Culicidae): Organogenesis.</a> Int. J. Insect morphol. &#38; Embryol. 7, 273-296 (1978).</li><li>Ivanova-Kazas, O. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+development+of+Anopheles+maculipennis+Mg.">Embryonic development of Anopheles maculipennis Mg.</a> Izv. Akad. Nauk SSSR ser. Biol. 2, 140-170 (1949).</li><li>Goltsev, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+and+evolutionary+basis+for+drought+tolerance+of+the+Anopheles+gambiae+embryo.">Developmental and evolutionary basis for drought tolerance of the Anopheles gambiae embryo.</a> Dev. Biol. 330, 462-470 (2009).</li><li>Papatsenko, D., Levine, M., Goltsev, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clusters+of+temporal+discordances+reveal+distinct+embryonic+patterning+mechanisms+in+Drosophila+and+Anopheles.">Clusters of temporal discordances reveal distinct embryonic patterning mechanisms in Drosophila and Anopheles.</a> PLoS Biol. 9, e1000584 (2011).</li><li>Davis, C. W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+of+larval+embryogenesis+in+the+mosquito+Culex+fatigans+Wiedemann+(Diptera:+Culicidae)+and+the+sheep-fly+Lucilla+seriata+Meigen+(Diptera:+Calliphoridae)+I.+Description+of+embryonic+development.">A comparative study of larval embryogenesis in the mosquito Culex fatigans Wiedemann (Diptera: Culicidae) and the sheep-fly Lucilla seriata Meigen (Diptera: Calliphoridae) I. Description of embryonic development.</a> Aust. J. Zool. 15, 547-579 (1967).</li></ol>

Yazarlar, herhangi bir açıklamaları var.

Situ ve kolorimetrik boyama protokolünde hibridizasyon tüm montaj sivrisinek doku ve embriyolar için buraya sunulan belirli organ ve hücre tipleri içinde transkript lokalizasyonu için yararlı bir tekniktir. Bu prosedürler bizim daha önce bildirilen yöntemlere göre, hem geniş yıkama adımları hızlandırarak ve ek teknik bilgi ve reaktif kaynaklardan sağlayan bir iyileşme vardır. Deneyimlerimize göre, melezleme sinyallerin kolorimetrik tespiti floresans tabanlı algılama düzenleri göre melezleşme sinyali hassasiyet ve netlik açısından üstündür. Ayrıca kolorimetrik tespiti gereği otomatik floresan olan embriyolar, sinyal ayrımcılık ile ilgili sorunları circumvents. Düşük bolluk transkript hedeflenen zaman hibridizasyon sinyalleri tespit Sınırlamalar, meydana ve arka plan boyama açıktır. 65 hibridizasyon sıcaklığının artmasıyla ° C geri azaltmak için bulunmuşturzemin hibridizasyon sinyalleri, ama tek hedefi özgü RNA probları tasarımı için önerilen bir alternatifi değildir. Bu protokol tüm montaj tükürük Aedes aegypti bezler ve yumurtalıklar ve Anofel gambiae, Anofel stephensi, Ae embriyoların in situ hibridizasyon gerçekleştirmek için kullanılmıştır. aegypti ve Culex quinquefasciatus. Bu yöntem diğer sivrisinek dokulara uygulanan ve muhtemelen diğer böceklerin gelenler. Ayrıca, üç vektör sivrisinek, Ae embriyonik gelişim evreleme için ilk kez karşılaştırmalı kılavuzlar için derlenmiş. aegypti, An. gambiae ve Cx. fatigans. Gözlemler sağduyulu yetiştirme koşullarında bu üç türün belirli suşları için bildirilmiştir. Bu gelişme süresi tabii sivrisinek türlerinin farklı suşları için ve farklı yetiştirme koşullarında değişiklik dikkat etmek önemlidir. In situ ekinde hibridizasyonları ana çabaları devam edenSivrisinek ve diğer eklembacaklıların transcriptomes lyze ve bu organizmaların gen ifadesinin düzenlenmesinde daha iyi bir görüntü sağlayabilir.

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> 0.5 M EDTA </td><td> Ambion </td><td> AM9261 </td><td></td></tr><tr><td> 1M Tris-HCl </td><td> Ambion </td><td> AM9855G </td><td> pH 8.0 </td></tr><tr><td> 10X PBS </td><td> Ambion </td><td> AM9625 </td><td></td></tr><tr><td> 20X SSC </td><td> Ambion </td><td> AM9763 </td><td></td></tr><tr><td> 1.5 ml mikrofüj tüpler </td><td> Ambion </td><td> AM12400 </td><td> Örnekleri görselleştirilmesi yardımcıları; az standart tüplere göre daha opak </td></tr><tr><td> Deiyonize formamid </td><td> Ambion </td><td> AM9342 </td><td> 4 at Depolama ° C </td></tr><tr><td> DEPC su </td><td> Ambion </td><td> AM9932 </td><td></td></tr><tr><td> Proteinaz K </td><td> Ambion </td><td> AM2546 </td><td></td></tr><tr><td> % 5.25 Sodyum hipoklorit </td><td> Austin&#39;in </td><td></td><td> A-1 Marka </td></tr><tr><td> T3 RNA Polimeraz-Plus </td><td> Ambion </td><td> AM2733 </td><td> -20 ° C&#39;de depolanması </td></tr><tr><td> T7 RNA polimeraz </td><td> Ambion </td><td> AM2082 </td><td> -20 ° C&#39;de depolanması </td></tr><tr><td> % 95 Etanol </td><td> Fisher Scientific </td><td> AC61511-0040 </td><td></td></tr><tr><td> Fisherbrand atılabilir polietilen transferi pipetler </td><td> Fisher Scientific </td><td> 13-711-7M </td><td></td></tr><tr><td> % 37 Formaldehit </td><td> Fisher Scientific </td><td> F79-500 </td><td></td></tr><tr><td> HPLC-grade metanol </td><td> Fisher Scientific </td><td> A452-1 </td><td></td></tr><tr><td> Magnezyum klorür </td><td> Fisher Scientific </td><td> M87-500 </td><td></td></tr><tr><td> Mikroskop cam kapak </td><td> Fisher Scientific </td><td> 12-542A </td><td> 18 x18 mm </td></tr><tr><td> N-heptan </td><td> Fisher Scientific </td><td> H350-1 </td><td></td></tr><tr><td> P-ksilen </td><td> Fisher Scientific </td><td> O5082-500 </td><td></td></tr><tr><td> Pyrex 9-iyi Nokta Plaka </td><td> Fisher Scientific </td><td> 13-748B </td><td> 100x85 mm </td></tr><tr><td> Sodyum klorür </td><td> Fisher Scientific </td><td> AC32730-0025 </td><td></td></tr><tr><td> Sodyum hidroksit </td><td> Fisher Scientific </td><td> SS255-1 </td><td></td></tr><tr><td> SuperFrost / Plus mikroskop </td><td> Fisher Scientific </td><td> 12-550-15 </td><td> 25x75x1.0 mm </td></tr><tr><td> Davlyn Kırmızı Clear-liner peruk bant </td><td> Doğrudan Saç </td><td> KIRMIZI-75R12 </td><td> Poly / x 12 yd bant rulo Cilt temel malzeme 0.75 </td></tr><tr><td> One ile Sequening için TOPOTA Klonlama Takımı Top10 kimyasal yetkili E. Shot coli </td><td> Invitrogen </td><td> K457501 K457540 </td><td> 20 reaksiyonlar 40 reaksiyonlar </td></tr><tr><td> Sonicated somon sperm DNA </td><td> Invitrogen </td><td> 15632-011 </td><td> -20 ° C&#39;de depolanması </td></tr><tr><td> Anti-digoxigenin-AP Fab fragmanları </td><td> Roche Applied Science </td><td> 1093274 </td><td> 4 at Depolama ° C </td></tr><tr><td> DIG RNA etiketleme mix </td><td> Roche Applied Science </td><td> 1277073 </td><td> -20 ° C&#39;de depolanması </td></tr><tr><td> NBT / BCIP stok solüsyonu </td><td> Roche Applied Science </td><td> 1681451 </td><td> 4 at Depolama ° C </td></tr><tr><td> Batı Engelleme Reaktif </td><td> Roche Applied Science </td><td> 11921673001 </td><td> 4 at Depolama ° C </td></tr><tr><td> Saatilene Hitech polyester mesh (330,130) </td><td> Saati Yazdır </td><td> 330,34 UO PW </td><td> 330 Konuları / inç, 34 mikron iplik çapı, turuncu renkli </td></tr><tr><td> Gliserin </td><td> Sigma </td><td> G6279-1 </td><td> PBT% 70 </td></tr><tr><td> Heparin sodyum tuzu </td><td> Sigma </td><td> H3393 </td><td></td></tr><tr><td> Tween 20 </td><td> Sigma </td><td> P1379-500 </td><td></td></tr><tr><td> % 37 Formaldehit </td><td> Ted Pella </td><td> 18508 </td><td> Amber ampul içinde 10 ml hacme </td></tr><tr><td> 16 oz. Kapaklı Yalnız Kağıt konteynerleri </td><td> Paper Company </td><td> SOLOKH16AJ8000 </td><td></td></tr><tr><td> Borosilikat cam scintillatbekâr vidalı kapaklı iyon flakon </td><td> VWR International </td><td> 66022-128 </td><td> 500, 20 ml durumda </td></tr><tr><td> Mühürlü Balonlu şal hücresel yastıklama malzemesi </td><td> VWR International </td><td> 500018-081 </td><td> 10 / ayak rulo 0,188 inç kalınlığında </td></tr><tr><td> Tavuk serumu </td><td></td><td></td><td> Tam kan ya kanat damardan ya da bir yavru tavuktan kalp ponksiyonu ile toplanır edildi. Kan soğutulmuş ve daha sonra 30 dakika süreyle buz üzerinde yerleştirilmiş kadar 1 saat 37 ° C&#39;de inkübe edildi. Serum 10 dakika boyunca 3000 x g&#39;de toplandı ve santrifüje edildi. Oluşan yüzey maddesi (serum açıklık), kullanılıncaya kadar toplandı ve -20 ° C&#39;de muhafaza edildi. </td></tr></tbody></table> Tablo 4. In situ melezleştirme için spesifik reaktifler ve ekipman Tablo.

hybridization in situ of salivary glands, ovaries, and embryos of vector mosquitoes

Sivrisinekler Arbovirüsler, protozoon parazit ve nematod gibi patojenlerin çeşitli bir dizi için bir vektör. Üreme yer sivrisinek host ve patojen etkileşim ve organlarda sivrisinek yoluyla bulaşan hastalık bulaşmasını azaltmak ve yumurta gelişimini bozabilir stratejileri için büyük ilgi oldugu dokularda ifade edilir transkript ve gen düzenleyici Araştırılması. Araçların bir dizi gen ekspresyonu zamansal ve doku-spesifik düzenlemeler incelemek ve onaylamak için istihdam edilmiştir. Burada, belirli genlerin ifade ve ürünleri birikir nerede anlayışımızı geliştiren mekansal bilgi elde etmek için geliştirilen protokoller açıklanmaktadır. Açıklanan protokolü yeniden, hangi ifadeyi doğrulamak ve bu kadın tükürük bezlerinin yanı sıra yumurtalık ve embriyoların Subselüler bölmeleri gibi sivrisinek kaynaklı patojen iletimi ile ilgili dokularında transkript birikimi desenleri belirlemek için kullanılmıştırsivrisinek üreme ve gelişme için geç. Aşağıdaki işlemleri hedef spesifik hibridizasyon sinyalleri kaybı olmadan protokolü çeşitli adımlardan etkinliğini arttırır iyileştirilmiş bir yöntem temsil eder. Toplanması, tespiti, ön hibridizasyon ve sivrisinek embriyoların hibridizasyon için belirli adımlar Bölüm B. ayrıntılı iken RNA prob hazırlanması için rehber, yumuşak doku ve fiksasyon ve hibridizasyon için genel prosedürü diseksiyonu, Bölüm A anlatılan

Watch this Scientific Journal Video about Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes at JoVE.com

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

Zamansal ve mekansal gen ekspresyon analizleri fonksiyonel genomik önemli bir role sahiptir. Tüm montaj hibridizasyon<em&gt; In situ</em&gt; Doku ve hücre içi bölmeler içinde transkriptlerin Lokalizasyon belirlemek açısından faydalıdır. Burada bir hibridizasyon ana hatlarıyla<em&gt; In situ</emSivrisinekler belirli hedef dokulara değişikliklerle&gt; protokolü.

Melezleme<em&gt; In situ</em&gt; Bezler, Yumurtalık ve Vektör Sivrisinek ve Embriyoların

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

hybridization-situ-salivary-glands-ovaries-embryos-vector

Research

JoVE Journal

Immunology and Infection

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

16.5K Görüntüleme.  University of California, Irvine. Zamansal ve mekansal gen ekspresyon analizleri fonksiyonel genomik önemli bir role sahiptir. Tüm montaj hibridizasyon In situ Doku ve hücre içi bölmeler içinde transkriptlerin Lokalizasyon belirlemek açısından faydalıdır. Burada bir hibridizasyon ana hatlarıyla In situ protokolü.

Video: Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector 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).

Yapışkan bant tabanlı Örnekleme ve Floresans kombinasyonu In situ</emHızlı Algılama> Hibridizasyon Salmonella Taze üretin

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

İmmünoloji ve Enfeksiyon

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes 1. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions 2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes 4, 5, 6. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti 7 and Cx. quinquefasciatus 8, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates 9. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti 10, the accumulation of multiple chromosomal rearrangements in cell line chromosomes 11 makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol 12 is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Among the three mosquito genera, namely Anopheles, Aedes, and Culex, physical genome mapping techniques were established only for Anopheles, whose members possess readable polytene chromosomes. For the genera of Aedes and Culex, however, cytogenetic mapping remains challenging because of the poor quality of polytene chromosomes. Here we present a universal protocol for obtaining high-quality preparations of mitotic chromosomes and an optimized FISH protocol for all three genera of mosquitoes.

Floresan<em&gt; In situ</emSivrisinekler ve Mitotik Kromozomlar üzerinde&gt; Hibridizasyon

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

Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection

Mosquito-borne viruses (MBVs), which are infectious pathogens to vertebrates, are spread by many mosquito species, posing a severe threat to public health. Once ingested, the viruses must overcome the mosquito midgut barrier to reach the hemolymph, from where they might potentially spread to the salivary glands. When a mosquito bites, these viruses are spread to new vertebrate hosts. Similarly, the mosquito may pick up different viruses. In general, only a tiny portion of viruses may enter the salivary glands via the gut. The transmission efficiency of these viruses to the glands is be affected by the two physical barriers found in different mosquito species: midgut barriers and salivary glands barriers. This protocol presents a method for virus detection in salivary glands of Aedes aegypti&#39;s&#160;following oral feeding and intrathoracic injection infection. Furthermore, determining whether the guts and/or salivary glands hinder viral spread can aid in the risk assessments of MBVs transmitted by Aedes aegypti.

This methodology, which included oral feeding and intrathoracic injection infection, could effectively assess the influence of midgut and/or salivary gland barriers on arbovirus infection.

Yetişkin Sivrisineklerde Oral Beslenme ve Mikroenjeksiyon ile Deneysel Viral Enfeksiyon

Mosquito-borne viruses (MBVs), which are infectious pathogens to vertebrates, are spread by many mosquito species, posing a severe threat to public ...

Sivrisinek Spermatogenezini İncelemek için Floresan In Situ Hibridizasyon

Whole-Mount Fluorescence In Situ Hybridization to Study Spermatogenesis in the Anopheles Mosquito

Spermatogenesis is a complex biological process during which diploid cells undergo successive mitotic and meiotic division followed by large structural changes to form haploid spermatozoa. Besides the biological aspect, studying spermatogenesis is of paramount importance for understanding and developing genetic technologies such as gene drive and synthetic sex ratio distorters, which, by altering Mendelian inheritance and the sperm sex ratio, respectively, could be used to control pest insect populations. These technologies have proven to be very promising in lab settings and could potentially be used to control wild populations of Anopheles mosquitoes, which are vectors of malaria. Due to the simplicity of the testis anatomy and their medical importance, Anopheles gambiae, a major malaria vector in sub-Saharan Africa, represents a good cytological model for studying spermatogenesis. This protocol describes how whole-mount fluorescence in situ hybridization (WFISH) can be used to study the dramatic changes in cell nuclear structure through spermatogenesis using fluorescent probes that specifically stain the X and Y chromosomes. FISH usually requires the disruption of the reproductive organs to expose mitotic or meiotic chromosomes and allow the staining of specific genomic regions with fluorescent probes. WFISH enables the preservation of the native cytological structure of the testis, coupled with a good level of signal detection from fluorescent probes targeting repetitive DNA sequences. This allows researchers to follow changes in the chromosomal behavior of cells undergoing meiosis along the structure of the organ, where each phase of the process can clearly be distinguished. This technique could be particularly useful for studying chromosome meiotic pairing and investigating the cytological phenotypes associated with, for example, synthetic sex ratio distorters, hybrid male sterility, and the knock-out of genes involved in spermatogenesis.

Yazar Spotlight: Anofel Sivrisineğinde Spermatogenezi İncelemek için Tam Montajlı Floresan In Situ Hibridizasyon  

Given their simple anatomy, Anopheles testes offer a good cytological model for studying spermatogenesis. This protocol describes whole-mount fluorescence in situ hybridization, a technique used to investigate this biological process, as well as the phenotype of transgenic strains harboring mutations in the genes involved in sperm production.

Anofel Sivrisineklerinde Spermatogenezi İncelemek için Tam Montajlı Floresan In Situ Hibridizasyon

Spermatogenesis is a complex biological process during which diploid cells undergo successive mitotic and meiotic division followed by large structural ...

Sivrisinek Koku Eklerinde Kemosensoriyel Gen Ekspresyon Haritalaması

Hybridization Chain Reaction RNA Whole-Mount Fluorescence In situ Hybridization of Chemosensory Genes in Mosquito Olfactory Appendages

Mosquitoes are effective vectors of deadly diseases and can navigate their chemical environment using chemosensory receptors expressed in their olfactory appendages. Understanding how chemosensory receptors are spatially organized in the peripheral olfactory appendages can offer insights into how odor is encoded in the mosquito olfactory system and inform new ways to combat the spread of mosquito-borne diseases. The emergence of third-generation hybridization chain reaction RNA whole-mount fluorescence in situ hybridization (HCR RNA WM-FISH) allows for spatial mapping and simultaneous expression profiling of multiple chemosensory genes. Here, we describe a stepwise approach for performing HCR RNA WM-FISH on the Anopheles mosquito antenna and maxillary palp. We investigated the sensitivity of this technique by examining the expression profile of ionotropic olfactory receptors. We asked if the HCR WM-FISH technique described was suitable for multiplexed studies by tethering RNA probes to three spectrally distinct fluorophores. Results provided evidence that HCR RNA WM-FISH is robustly sensitive to simultaneously detect multiple chemosensory genes in the antenna and maxillary palp olfactory appendages. Further investigations attest to the suitability of HCR WM-FISH for co-expression profiling of double and triple RNA targets. This technique, when applied with modifications, could be adaptable to localize genes of interest in the olfactory tissues of other insect species or in other appendages.

Yazar Spotlight: Sivrisineklerde Koku Reseptör İfadesini Görselleştirme 

The article describes the methods and reagents necessary to perform hybridization chain reaction RNA whole-mount fluorescence in situ hybridization (HCR RNA WM-FISH) to reveal insights into the spatial and cellular resolution of chemosensory receptor genes in the mosquito antenna and maxillary palp.

Hibridizasyon Zincir Reaksiyonu RNA Tam Montajlı Floresan Sivrisinek Koku Alma Eklerinde Kemosensoriyel Genlerin In situ Hibridizasyonu,

Mosquitoes are effective vectors of deadly diseases and can navigate their chemical environment using chemosensory receptors expressed in their olfactory ...

Nörobilim

Forced Salivation As a Method to Analyze Vector Competence of Mosquitoes

Vector competence is defined as the potential of a mosquito species to transmit a mosquito-borne virus (mobovirus) to a vertebrate host. Viable virus particles are transmitted during a blood meal via the saliva of an infected mosquito. Forced salivation assays allow determining the vector potential on the basis of single mosquitoes, avoiding the use of animal experiments. The method is suitable to analyze a large number of mosquitoes in one experiment within a short period of time. Forced salivation assays were used to analyze 856 individual mosquitoes trapped in Germany, including two different Culex pipiens pipiens biotypes, Culex torrentium as well as Aedes albopictus, which were experimentally infected with Zika virus (ZIKV) and incubated at 18 &#176;C or 27 &#176;C for two and three weeks. The results indicated the lack of vector competence of the different Culex taxa for ZIKV. In contrast, Aedes albopictus was susceptible to ZIKV, but only at 27 &#176;C, with transmission rates similar to an Aedes aegypti laboratory colony tested in parallel.

For efficient control of mosquito borne virus transmission, the knowledge of the vector potential of respective mosquitoes is of particular interest. We describe forced salivation as a method to analyze vector competence of Aedes albopictus and three different Culex taxa for the transmission of Zika virus.

Salya vektör yetkinlik sivrisinek analiz etmek için bir yöntem olarak zorla

Vector competence is defined as the potential of a mosquito species to transmit a mosquito-borne virus (mobovirus) to a vertebrate host. Viable virus ...

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Anofel Sivrisineklerinde Spermatogenezi İncelemek için Tam Montajlı Floresan In Situ Hibridizasyon

Anofel Sivrisineklerinde Spermatogenezi İncelemek için Tam Montajlı Floresan In Situ Hibridizasyon

Anofel Sivrisineklerinde Spermatogenezi İncelemek için Tam Montajlı Floresan In Situ Hibridizasyon

Hibridizasyon Zincir Reaksiyonu RNA Tam Montajlı Floresan Sivrisinek Koku Alma Eklerinde Kemosensoriyel Genlerin In situ Hibridizasyonu,

Hibridizasyon Zincir Reaksiyonu RNA Tam Montajlı Floresan Sivrisinek Koku Alma Eklerinde Kemosensoriyel Genlerin In situ Hibridizasyonu,

Hibridizasyon Zincir Reaksiyonu RNA Tam Montajlı Floresan Sivrisinek Koku Alma Eklerinde Kemosensoriyel Genlerin In situ Hibridizasyonu,

Salya vektör yetkinlik sivrisinek analiz etmek için bir yöntem olarak zorla