著者らは、その後にハイブリダイゼーションのためにここで記述されたプロトコルを開発するために適応し、変更された、 ハマダラカの胚の in situ ハイブリダイゼーションのためのプロトコルの議論のための軟組織とユーリGoltsevその場の方法でハイブリダイゼーションの開発に助言をマリカ·ウォルターズに感謝したいと思いますヒトスジシマカとアカイエカの胚の in situ。我々はまた、アダム·パレとDavid K​​osmanによって与えられた有用な提言を認める。我々は科学的な議論とプロトコルのテキストを編集するためのオズワルドMarinottiに感謝します。

<ol>
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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>

著者らは開示しません。

全体のマウント蚊組織および胚のためにここで紹介する場と比色染色プロトコールでのハイブリダイゼーションは、特定の臓器や細胞型内転写産物の局在のために有用な技術である。これらの手順は、私たちの以前に報告された方法に比べて、両方の広範な洗浄工程の合理化と技術的な詳細および試薬ソースを提供することで改善されています。 我々の経験では、ハイブリダイゼーションシグナルの比色検出は、蛍光ベースの検出方式に比べてハイブリダイゼーションシグナルの感度と透明性に優れています。また、比色検出は、本質的に自動蛍光性である胚の信号差別に関連する問題を回避します。低濃度の転写産物をターゲットにされている場合ハイブリダイゼーションシグナルを検出するのに制限が発生し、バックグラウンド染色は明白である。 65ハイブリダイゼーション温度を上昇させる°Cは、バック減らすことが見出されているグラウンドハイブリダイゼーションシグナルが、固有のターゲットに特異的なRNAプローブを設計するための提案代わるものではありません。 このプロトコルは、全マウント唾液ネッタイシマカの腺、卵巣、 ハマダラカ、ハマダラカ、AEの胚の in situ ハイブリダイゼーションを行うために使用されています。ネッタイシマカとネッタイイエカ 。また、このメソッドは、他の蚊の組織に適用されると、おそらく他の昆虫のものです。さらに、我々は3つのベクトル蚊、 ヒトスジシマカにおける胚発生のステージングのための最初の時間の比較のガイドラインについては、コンパイルされます。ネッタイシマカ、。ハマダラカとCx。 fatigans。観測は控えめな飼育条件下では、これら3種の特定の株が報告されている。開発時間のコースは蚊種の異なる株のために、異なる飼育条件下で異なることに注意することが重要です。 ANAへの努力継続的にその場サプリメントでハイブリダイゼーション蚊やその他の節足動物のトランスクリプトームをlyze、これらの生物における遺伝子発現の調節の良い画像を提供することがあります。

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td> 0.5 M EDTA </td><td> Ambionの</td><td> AM9261 </td><td></td></tr><tr><td> 1MのTris-HCl </td><td> Ambionの</td><td> AM9855G </td><td> pH8.0 </td></tr><tr><td> 10×PBS </td><td> Ambionの</td><td> AM9625 </td><td></td></tr><tr><td> 20X SSC </td><td> Ambionの</td><td> AM9763 </td><td></td></tr><tr><td> 1.5mlマイクロチューブ</td><td> Ambionの</td><td> AM12400 </td><td>可視化サンプル中のエイズ、標準のチューブよりも小さい不透明な</td></tr><tr><td>脱イオン化ホルムアミド</td><td> Ambionの</td><td> AM9342 </td><td> 4°Cでの保存</td></tr><tr><td> DEPC水</td><td> Ambionの</td><td> AM9932 </td><td></td></tr><tr><td>プロテイナーゼK </td><td> Ambionの</td><td> AM2546 </td><td></td></tr><tr><td> 5.25％次亜塩素酸ナトリウム</td><td>オースティンの</td><td></td><td> A-1ブランド</td></tr><tr><td> T3 RNAポリメラーゼプラス</td><td> Ambionの</td><td> AM2733 </td><td> -20°Cでの貯蔵</td></tr><tr><td> T7 RNAポリメラーゼ</td><td> Ambionの</td><td> AM2082 </td><td> -20°Cでの貯蔵</td></tr><tr><td> 95％エタノール</td><td>フィッシャー·サイエンティフィック</td><td> AC61511-0040 </td><td></td></tr><tr><td> Fisherbrand使い捨てポリエチレン転送ピペット</td><td>フィッシャー·サイエンティフィック</td><td> 13から711-7M </td><td></td></tr><tr><td> 37％ホルムアルデヒド</td><td>フィッシャー·サイエンティフィック</td><td> F79-500 </td><td></td></tr><tr><td> HPLCグレードのメタノール</td><td>フィッシャー·サイエンティフィック</td><td> A452-1 </td><td></td></tr><tr><td>塩化マグネシウム</td><td>フィッシャー·サイエンティフィック</td><td> M87-500 </td><td></td></tr><tr><td>顕微鏡のカバーガラス</td><td>フィッシャー·サイエンティフィック</td><td> 12-542A </td><td> 18×18ミリメートル</td></tr><tr><td> n-ヘプタン</td><td>フィッシャー·サイエンティフィック</td><td> H350-1 </td><td></td></tr><tr><td> P-キシレン</td><td>フィッシャー·サイエンティフィック</td><td> O5082-500 </td><td></td></tr><tr><td>パイレックス9ウェルスポットプレート</td><td>フィッシャー·サイエンティフィック</td><td> 13-748B </td><td> 100x85ミリメートル</td></tr><tr><td>塩化ナトリウム</td><td>フィッシャー·サイエンティフィック</td><td> AC32730-0025 </td><td></td></tr><tr><td>水酸化ナトリウム</td><td>フィッシャー·サイエンティフィック</td><td> SS255-1 </td><td></td></tr><tr><td> Superfrost /プラス顕微鏡スライド</td><td>フィッシャー·サイエンティフィック</td><td> 12-550-15 </td><td> 25x75X1.0ミリメートル</td></tr><tr><td> Davlynレッドクリアライナーかつらテープ</td><td>直接髪</td><td> RED-75R12 </td><td>ポリ/ X 12ヤードテープロールのスキン基材0.75 </td></tr><tr><td>ワントップ10化学的にコンピテントE.とSequeningためのTOPOTAクローニングキット大腸菌 </td><td>インビトロジェン</td><td> K457501 K457540 </td><td> 20反応 40反応</td></tr><tr><td>超音波処理サケ精子DNA </td><td>インビトロジェン</td><td> 15632-011 </td><td> -20°Cでの貯蔵</td></tr><tr><td>抗ジゴキシゲニン-AP、Fabフラグメント</td><td>ロシュ·ダイアグノスティックス</td><td> 1093274 </td><td> 4°Cでの保存</td></tr><tr><td> DIG RNAラベリングミックス</td><td>ロシュ·ダイアグノスティックス</td><td> 1277073 </td><td> -20°Cでの貯蔵</td></tr><tr><td> NBT / BCIPストック溶液</td><td>ロシュ·ダイアグノスティックス</td><td> 1681451 </td><td> 4°Cでの保存</td></tr><tr><td>西部のブロッキング試薬</td><td>ロシュ·ダイアグノスティックス</td><td> 11921673001 </td><td> 4°Cでの保存</td></tr><tr><td> Saatileneハイテックポリエステルメッシュ（330.130） </td><td> Saatiプリント</td><td> 330.34 UO PW </td><td> 330スレッド/インチ、34ミクロンのネジ径、オレンジ色</td></tr><tr><td>グリセロール</td><td>シグマ</td><td> G6279-1 </td><td> PBTの70％ </td></tr><tr><td>ヘパリンナトリウム塩</td><td>シグマ</td><td> H3393 </td><td></td></tr><tr><td> Tween 20を</td><td>シグマ</td><td> P1379-500 </td><td></td></tr><tr><td> 37％ホルムアルデヒド</td><td>テッド·ペラ</td><td> 18508 </td><td>琥珀色のアンプルに10 mlのアリコート</td></tr><tr><td> 16オンス蓋つきのソロ紙容器</td><td>ペーパーカンパニー</td><td> SOLOKH16AJ8000 </td><td></td></tr><tr><td>ホウケイ酸ガラスscintillatアタッチされていないスクリューキャップ付きバイアルイオン</td><td> VWRインターナショナル</td><td> 66022-128 </td><td> 500の20ミリリットルの場合</td></tr><tr><td>シールドエアーバブルラップcelluar緩衝材</td><td> VWRインターナショナル</td><td> 500018-081 </td><td> 10フィート/ロール0.188インチの厚さ</td></tr><tr><td>鶏血清</td><td></td><td></td><td>全血は、どちらの翼静脈から、または幼若鶏から心臓穿刺により採取した。血液が凝固して、30分間氷上に置いたまでは1時間37℃でインキュベートした。血清を10分間3000×gで収集し、遠心分離した。得られた上清（明らかに血清）を使用するまで-20℃で収集し、格納されていました。 </td></tr></tbody></table> 表4。in situでのハイブリダイゼーションの特異的試薬および機器の表。

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

蚊はアルボウイルス、原虫や線虫を含む病原体の多様なセットのベクトルです。蚊の宿主と病原体相互作用の組織で発現し、臓器に繁殖に関与している転写産物や遺伝子レギュレータの調査では、戦略は蚊が媒介する病気の感染を減らすと卵の開発を混乱させるための大きな関心である。ツールの数は、遺伝子発現の時間的および組織特異的調節を研究し、検証するために用いられてきた。ここでは、特定の遺伝子が発現し、それらの製品が蓄積されている場所の我々の理解を高め、空間情報を取得するために開発されているプロトコルについて説明します。記述されているプロトコルは、再され、式を検証し、そのような女性の唾液腺と同様に、卵巣および胚の細胞内コンパートメントなど蚊が媒介する病原体の伝播に関連した組織では転写産物の蓄積パターンを決定するために使用されています蚊の再生と発展に遅い。 次の手順では、ターゲット固有のハイブリダイゼーションシグナルを失わずにプロトコルのさまざまなステップの効率を向上させる最適化された方法論を表しています。 RNAプローブの調製のためのガイドライン、軟組織と固定とハイブリダイゼーションのための一般的な手順の解剖は、蚊の胚の収集、固定、プレハイブリダイゼーションおよびハイブリダイゼーションのための具体的な手順は、パートBに記載されていながら、パートで説明されています

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

時間的·空間的遺伝子発現解析は、ゲノム機能解析において重要な役割を持っています。ホールマウントハイブリダイゼーション<em&gt;その場で</em&gt;組織や細胞内コンパートメント内での転写産物の局在を決定するために有用である。ここでは、ハイブリダイゼーションを概説<em&gt;その場で</em蚊の特定の標的組織のために修正を加えた&gt;プロトコル。

ハイブリダイゼーション<em&gt;その場で</em&gt;唾液腺、卵巣、および媒介蚊の胚の

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 ビュー.  University of California, Irvine. 時間的·空間的遺伝子発現解析は、ゲノム機能解析において重要な役割を持っています。ホールマウントハイブリダイゼーションその場で組織や細胞内コンパートメント内での転写産物の局在を決定するために有用である。ここでは、ハイブリダイゼーションを概説その場でプロトコル。

ビデオ: 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).

粘着テープベースのサンプリングと蛍光の組み合わせその場で</emの迅速検出のための>ハイブリダイゼーションサルモネラ生鮮食品について

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

免疫・感染学

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

Vero細胞培養アッセイによるフィールドコレクト蚊から感染性ウイルスの検出

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

マダニScapularisダニの唾液、唾液腺、および体液コレクション

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

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.

蛍光灯<em&gt;その場で</em蚊の分裂期染色体上&gt;ハイブリダイゼーション

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

A Method for the Measurement of Salivary Gland Function in Mice

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Salivary gland hypofunction is a frequent consequence of autoimmune disease and radiation therapy. Reproducible evaluation of salivary gland function in mouse models of these diseases is a technical challenge. Here, a simple method for accurate and reproducible measurement of saliva production in mice is described.

マウスの唾液腺機能の測定法

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...

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).

KSHV感染細胞における特定遺伝子産物の高量蛍光ハイブリダイゼーション(FISH)と免疫蛍光(IF)における定量蛍光

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

Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to restore function of glands damaged by radiation therapies or due to pathologies associated with Sj&#246;gren's syndrome. A second goal of researchers is to define the mechanisms by which viruses replicate within glandular tissue where they can then gain access to salivary fluids important for horizontal transmission. These goals highlight the need for a robust and accessible in vitro salivary gland model that can be utilized by researchers interested in the above mentioned as well as related research areas. Here we discuss a simple protocol to isolate epithelial cells from human salivary glands and propagate them in vitro. Our protocol can be further optimized to meet the needs of individual studies. Briefly, salivary tissue is mechanically and enzymatically separated to isolate single cells or small clusters of cells. Selection for epithelial cells occurs by plating onto a basement membrane matrix in the presence of media optimized to promote epithelial cell growth. These resulting cultures can be maintained as three-dimensional clusters, termed "salispheres", or grown as a monolayer on treated plastic tissue culture dishes. This protocol results in the outgrowth of a heterogenous population of mainly epithelial cells that can be propagated for 5-8 passages (15-20 population doublings) before undergoing cellular senescence.

We present a method for isolating and cultivating primary human salivary gland-derived epithelial cells. These cells exhibit gene expression patterns consistent with them being of salivary epithelial origin and can be grown as salispheres on basement membrane matrices derived from Engelbreth-Holm-Swarm tumor cells or as monolayers on treated culture dishes.

唾液圏または単層としての体外増殖のためのヒト唾液腺からの唾液上皮細胞の単離

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to ...

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.

上皮内蛍光を用いたマウス消化管におけるカンダアルビカンスの可視化

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.

ジカウイルスを用いた Aedes aegypti蚊の ベクターコンピテンス分析

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

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.

SITUハイブリダイゼーションにおける免疫RNA-蛍光を用いたSARS-CoV-2の可視化

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

Methods Article

ハイブリダイゼーション