我々は、 ハマダラカで染色体の準備と魚と彼らの助けのためにセルゲイデーミンとタチアナKaramyshevaに感謝します。また、テキストを編集するために私達にヒトスジシマカとアカイエカゲノムDNA BACクローンとメリッサウェイドを提供するためのデイビッドSeversonに感謝します。イゴールV. SharakhovにマリアV. SharakhovaとAI094289 1R21-01 1R21 AI88035-01：この作品は、国立衛生研究所からの2の補助金によって支えられている。

<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. 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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. 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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>

特別な利害関係は宣言されません。

蚊の分裂期染色体上の非蛍光in situハイブリダイゼーションは、A.クマーとK.チェンライ17によって1990年に初めて行われた。その研究では、18Sと28SリボソームDNA遺伝子は、プラスミド1で一緒にクローニングされた蚊の20種の染色体に配置した。 DNAプローブを放射性標識し、脳の神経節からの染色体にハイブリダイズさせた。 3蚊属の中では、FISH法は、AEの細胞株から分裂期染色体のために開発されました。ライブ蚊から有糸分裂染色体上ネッタイシマカ 10,18,19および実行されたことがありません。最近、我々は第 4齢幼虫9のIDから、高品質な染色体の準備を取得するための、シンプルで堅牢な技術を開発しました。この方法では、染色体の数が多いが、1スライドに得ることを可能にすると普遍的に蚊のすべての種のために使用することができます。唯一の幼虫、蛹、大人ではないSTAを使用する必要蚊のGESは、スライドの準備のために、おそらく方法の唯一の制限事項です。標準FISH法12は ヒトスジシマカ、アカイエカ、およびハマダラカの分裂期染色体のプローブとしてゲノムBACクローンとIGS rDNAを用いて最適化されています。 これらの特定のアプリケーションに加えて、ここで説明したFISHプロトコルは、他の目的にも使用することができます。非特異的なハイブリダイゼーションを阻止するためのC 0トンのDNA画分を利用して高度なFISHプロトコルも、BACクローンまたはハマダラカのヘテロクロマチン領域における他の大規模なDNA断片のハイブリダイゼーションのために適用することができます。ヘテロクロマチン領域は転移因子や他のリピートを豊かにし、これらの地域からのプローブは通常、染色体3上の強力なバックグラウンドを生産している。ラベルなしのC 0トンのDNA画分を使用すると、染色体へのプローブの非特異的ハイブリダイゼーションを削減するのに役立ちます。 FISHのprのシンプルバージョンotocolは蚊や他の昆虫の分裂期染色体上の任意のrDNAまたは反復的なDNAプローブを使用することができます。さらに、それはまた、 蚊やショウジョウバエなどのユークロマチン領域における低反復DNA含量の種におけるBACクローンDNAのハイブリダイゼーションのために適用することができます。ここで提案されたプロトコルは、蚊の高度に完成した染色体ベースのゲノムアセンブリを取得するのに役立ちますし、広く昆虫の他のグループ内のさまざまな細胞遺伝学的用途に使用することができる。

<table border="1"><tbody><tr><td> 試薬/機器の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> 注釈 </td></tr><tr><td> MZ6ライカの実体顕微鏡</td><td>ライカ</td><td> VA-OM-E194-354 </td><td>異なった実体顕微鏡を使用することができます</td></tr><tr><td>オリンパスCX41位相差顕微鏡</td><td>オリンポス</td><td> CX41 </td><td>異なる位相差顕微鏡を使用することができます</td></tr><tr><td>オリンパスBX61蛍光顕微鏡</td><td>オリンポス</td><td> BX61 </td><td>異なる蛍光顕微鏡を使用することができます</td></tr><tr><td> ThermoBriteスライド変性/ハイブリダイゼーションシステム</td><td>アボット分子</td><td> 30-144110 </td><td>加熱ブロック多湿の室となる</td></tr><tr><td>解剖針</td><td>ファインScienceTools </td><td> 10130から10</td><td></td></tr><tr><td>ニードルホルダー</td><td>ファイン科学ツール</td><td> 26018から17 </td><td></td></tr><tr><td>解剖用はさみ</td><td>ファイン科学ツール</td><td> 15000から03 </td><td></td></tr><tr><td> 75x25ダブルすりガラスマイクロスライド</td><td>コー​​ニング</td><td> 2949-75x25 </td><td></td></tr><tr><td> 22x22のmmの顕微鏡カバースリップ</td><td>フィッシャー·サイエンティフィック</td><td> 12-544-10 </td><td></td></tr><tr><td>パラフィルム</td><td>フィッシャー·サイエンティフィック</td><td> 13-374-10 </td><td></td></tr><tr><td>ゴムのり</td><td>フィッシャー·サイエンティフィック</td><td> 50-949-105 </td><td></td></tr><tr><td>酢酸</td><td>フィッシャー·サイエンティフィック</td><td> A491-212 </td><td></td></tr><tr><td>アルコール200プルーフ</td><td>ディーラボラトリーズ</td><td> 2701 </td><td></td></tr><tr><td>プロピオン酸</td><td>シグマAldr白点病</td><td> 402907 </td><td></td></tr><tr><td>塩酸</td><td>フィッシャー·サイエンティフィック</td><td> A144-500 </td><td></td></tr><tr><td>クエン酸ナトリウム二水和物</td><td>フィッシャー·サイエンティフィック</td><td> S279-500 </td><td></td></tr><tr><td>酢酸ナトリウム三水和物</td><td>フィッシャー·サイエンティフィック</td><td> BP334-500 </td><td></td></tr><tr><td>塩化カリウム</td><td>フィッシャー·サイエンティフィック</td><td> BP366-500 </td><td></td></tr><tr><td> EDTAを</td><td>フィッシャー·サイエンティフィック</td><td> S311-500 </td><td></td></tr><tr><td>トリス塩基</td><td>フィッシャー·サイエンティフィック</td><td> BP152-1 </td><td></td></tr><tr><td> 10倍のPBS </td><td>インビトロジェン</td><td> P5493 </td><td></td></tr><tr><td> 10パーセントNBF（中性緩衝ホルマリン） </td><td>シグマアルドリッチ</td><td> HT501128 </td><td></td></tr><tr><td> 99％ホルムアミド</td><td>フィッシャーンスntific </td><td> BP227500 </td><td></td></tr><tr><td>デキストラン硫酸エステルナトリウム塩</td><td>シグマアルドリッチ</td><td> D8906 </td><td></td></tr><tr><td> 20×SSC緩衝液</td><td>インビトロジェン</td><td> AM9765 </td><td></td></tr><tr><td> 1mMのYOYO-1ヨウ（509分の491）ソリューション</td><td>インビトロジェン</td><td> Y3601 </td><td></td></tr><tr><td>ゴールド試薬を延長退色</td><td>インビトロジェン</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>は、dATP、dCTP、dGTP、dTTPの</td><td>フェルメンタス</td><td> R0141、R0151、R0161、R0171 </td><td></td></tr><tr><td>にCy3-dUTPを、Cy5で-dUTPを</td><td> GEヘルスケア</td><td> PA53022、PA55022 </td><td></td></tr><tr><td> BSA </td><td>シグマアルドリッチ</td><td> A3294 </td><td></td></tr><tr><td> DNAポリメラーゼI </td><td>フェルメンタス</td><td> EP0041 </td><td></td></tr><tr><td> DNase Iを</td><td>フェルメンタス</td><td> EN0521 </td><td></td></tr><tr><td> S1ヌクレアーゼ</td><td>フェルメンタス</td><td> EN0321 </td><td></td></tr><tr><td> Taq DNAポリメラーゼ</td><td>インビトロジェン</td><td> 18038-042 </td><td></td></tr><tr><td>アーゼ</td><td>シグマアルドリッチ</td><td> 9001-99-4 </td><td></td></tr><tr><td>ペプシン</td><td> USB </td><td> 9001-75-6 </td><td></td></tr><tr><td>サケ精子DNA </td><td>シグマアルドリッチ</td><td> D7656 </td><td></td></tr><tr><td>ノニデット-P40（NP40） </td><td>米国の生物</td><td> NC9375914 </td><td></td></tr><tr><td>キアゲン血液および細胞培養Maxikit </td><td>キアゲン</td><td> 13362 </td><td></td></tr><tr><td>キアゲン大コンストラクトキット</td><td>キアゲン</td><td> 12462 </tD&gt; <td></td></tr></tbody></table>

fluorescent in situ hybridization on mitotic chromosomes of mosquitoes

in situハイブリダイゼーション（FISH） 蛍光 inは、日常的にDNAとRNAプローブの染色体上の位置を決定するために、多くの研究室で使用されるテクニックです。この方法の一つの重要なアプリケーションは、様々な生物のゲノムアセンブリを改善するのに有用で高品質な物理マップの開発である。多糸と糸分裂染色体の自然なバンドパターンは、ゲノムsupercontigsの正確な順序およびオリエンテーションのためのガイダンスを提供します。すなわち3蚊属、 ハマダラカ 、 ヒトスジシマカ、アカイエカとの間で、十分に確立された染色体ベースのマッピング技術は、そのメンバーだけが読める多糸染色体1を所有ハマダラカ 、ために開発されました。ゲノムマッピングの努力の結果として、全体の88％。ハマダラカのゲノムは、正確な染色体位置2,3に配置されています。他の二つの蚊属、 ヤブカとアカイエカは、不十分な染色体bをpolytenizedたそれらのゲノム4、5、6の転移因子の重要overrepresentationのecause。ゲノムsupercontingsは31、9％ は 、AEの染色体への順序や向きをせずに割り当てられています。ネッタイシマカ7とそれぞれネッタイイエカ 8。これら2種の有糸分裂染色体の準備は以前に脳基底核および細胞株に限られていた。しかし、蚊の脳の神経節から調製した染色体のスライドは通常中期プレート9の低い数字が含まれています。また、FISH法は、AEの細胞株から分裂期染色体のために開発されてきたが。ネッタイシマカ 10は 、細胞株の染色体11内に複数の染色体再配列の蓄積は、ゲノムマッピングのためにそれらは無用になります。ここでは、C 4 番目の幼虫の成虫から高品質の分裂期染色体標本を得るためのシンプルで堅牢な技術（ID）を記述する蚊のすべての3つの属にも使用できます。標準FISHプロトコル12は AEの有糸分裂染色体上のプローブとしてゲノムDNAのBACクローンを使用するために最適化されています。ネッタイシマカとネッタイイエカ、オンプローブとしてリボソームDNAの遺伝子間スペーサー（IGS）の領域（遺伝子組換え）を利用する。ハマダラカの染色体は、物理マッピングに加えて、開発された技術は、人口細胞遺伝学、染色体分類/蚊や他の昆虫群の系統に適用することができます。

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

3蚊属のうち、すなわち<em&gt;ハマダラカ、</em&gt;<em&gt;ヤブカ</em&gt;と、<em&gt;イエカ</em&gt;物理的なゲノムマッピング技術が確立されたのみ<em&gt;ハマダラカ</em&gt;、そのメンバーは、読み取り可能な多糸染色体を持っています。の世代のために<em&gt;ヤブカ</em&gt;と<em&gt;イエカ</em&gt;しかし、細胞遺伝学的マッピングがあるため多糸染色体の質が悪く依然として厳しい。ここでは、高品質の有糸分裂染色体の準備と蚊のすべての3つの属のために最適化されたFISHプロトコルを取得するための汎用プロトコルを提示します。

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. ...

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Immunology and Infection

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

25.6K ビュー.  Virginia Tech. 3蚊属のうち、すなわちハマダラカ、ヤブカと、イエカ物理的なゲノムマッピング技術が確立されたのみハマダラカ、そのメンバーは、読み取り可能な多糸染色体を持っています。の世代のためにヤブカとイエカしかし、細胞遺伝学的マッピングがあるため多糸染色体の質が悪く依然とし?...

ビデオ: 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).

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

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.

ロックされた核酸のフローサイトメトリー、蛍光その場でハイブリダイゼーション（LNAフロー- FISH）：細菌のsmall RNAの検出のための方法

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.

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 ...

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

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

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

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

視覚化し、定量化するために蛍光タンパク質を使用しました<em&gt;クラミジア</em生細胞内&gt;液胞の成長ダイナミクス

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

2D and 3D Chromosome Painting in Malaria Mosquitoes

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

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

マラリア蚊の2Dと3D染色体絵画

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

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

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

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

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

体内上部気道免疫メディエーター レベルの定量化のための粘膜内層流体の非侵襲的サンプリング

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

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

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

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

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

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 ...

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 ...

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.

アエデスアエジプティ蚊における微小胞体寄生虫・エダリア・アダイスの伝播

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