A.は、C6/36細胞株にウイルスを伝播します。 <ol><li>細胞は75 cm 2でフラスコに80％コンフルエントに成長する。 </li><li>フラスコ内に残っ​​ている3〜4ミリリットルで、メディアを削除する。 </li><li> 0.5mlの株式のウイルスを取り出し、細胞あたり1.5ウイルス粒子の感染多重度で、上記のセルに追加します。徐々に室温で15分間フラスコを振とう。 </li><li> 5％CO 2、37℃で45分間インキュベート</li><li> 30mlのメディアを追加し、5日間インキュベートする。 </li></ol> B.は、ウイルスと血液の混合物を準備する<ol><li>スクレーパーで細胞をデタッチし、50 mLコニカルチューブに細胞とメディアを転送する。 </li><li>上清を収集するが、細胞ペレットと上清1mlのまま、10分間、800グラムでCentrifugate。 </li><li> ℃の水浴、繰り返し2〜3倍の37に解凍し乾燥したCO 2に、上記の細胞ペレットを凍結し、 </li><li>上清を取り、ステップ3から採取した上清と結合して、10分間800グラムでCentrifugate。 </li><li>熱帯熱マラリア原虫を持つハマダラカ蚊を感染させるために生殖母細胞培養の調製のための同じ手順（ステップ3）でのヒト全血を集める。 </li><li>ステップ4からウイルス上清を上記のヒト全血の同量を組み合わせて、ヒト血清を（全体の体積の10％）を追加。 </li><li> 37℃の水浴で30分間ヒト血液およびデングウイルスの上記混合物をインキュベート</li></ol> C.の餌付けの蚊この手順では、蚊の熱帯熱マラリア原虫感染症のセクションで説明されているものとほとんど同じです。吸血後14日目7日目、および唾液感染で我々は、アッセイ中腸感染症を。ウイルスと接触して来たすべての材料は、75％エタノールで、次に10％の漂白剤との最初の扱われます。 D.アッセイ蚊組織におけるウイルス力価<ol><li>二、三日蚊の組織解剖前に、細胞はアッセイが行われる日で80％コンフルエントに達するように24ウェルプレートでのC6/36細胞を成長させる。 </li><li>蚊は4で麻酔℃で1分間75％エタノールでそれらを浸すことによって犠牲と滅菌表面C、。その後、蚊は、解剖顕微鏡下で腸や唾液腺の解剖前に二度滅菌水で洗浄した。このステップからは、以下のすべての手順は、このような生物学的安全キャビネットのような無菌環境で実施する必要があります。これらの組織は、150μlの培地を含む試験管に移し、90秒間Kontesペレット乳棒モーターでホモジナイズされています。 </li><li> 990μLのメディアにホモジネートの10μlを添加することにより100倍希釈液を加えます。 </li><li>さらに1を確認：10、1:100と96ウェルプレートの培地で1:1000希釈; </li><li> 24ウェルプレートでメディアを取り出し、それぞれの対応するウェルに上記の4つの希釈液の各々の100μlを加える。 </li><li>その後、5％で45分間インキュベートし、室温で15分間ゆっくりプレートを振るCO 2、37 ° C; </li><li>各ウェルにmethycelluloseオーバーレイ（1％）の1mlを追加します。 </li><li> 37プレートをインキュベート℃で5日間、5％CO 2で。 </li><li>ここから無菌環境を必要としない上に、任意の他の病原体のケアと同様のステップは常に注意が必要です。各プレートからmethycelluloseのオーバーレイを破棄し、余分なメディアを削除するには汚点</li><li>各ウェルにメタノール/アセトン固定液（1:1）を1mlを追加。 1時間4℃でおいてください。 </li><li>固定液を捨て、そして1X PBSで1回洗浄する。 </li><li> 5パーセントへべれけとウイルスに対する一次抗体に1:1000希釈を行う。 </li><li> 37℃でインキュベート、各ウェルに希釈した高度免疫液200μlを加え℃で1時間のためのC; </li><li>高度免疫体液を削除し、1 × PBSでプレートを洗浄</li><li> 5パーセントへべれけ（1 X PBSを100mlの5グラム脱脂粉乳）でヤギ抗マウス共役（KPLカタログ番号074〜1806）の1:1000希釈を準備し、各ウェルに200μlを加える、とでインキュベート1時間37 ° C; </li><li>次のように基板を準備する：溶解後、1 X PBSの20ml中の3&#39; -ジアミノベンジジンterrahydrochloride（Sigmaカタログ＃D5905）の1錠を追加し、次に30％過酸化水素ペルオキシダーゼの8μlを追加する</li><li>各ウェルに上記基板の200μlを追加。 10分間室温で放置</li><li>基板を取り外し、各ウェルに蒸留水を1mlを加えて反応を停止</li><li>乾燥して水とブロットプレートを捨て、 </li><li>ウイルス粒子を数える</li></ol>

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Incubator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>with 5% CO2, 37oC</td>
</tr>
<tr>
<td>50 mL conical tubes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>plastic</td>
</tr>
<tr>
<td>human serum and blood</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>O+</td>
</tr>
<tr>
<td>pipette tip</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>for tissue culture </td>
</tr>
<tr>
<td>pipettman</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pasteur pipetts</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>glass, sterile</td>
</tr>
<tr>
<td>water bath</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>37C</td>
</tr>
<tr>
<td>centrifuge</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>parafilm</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>circulating water bath</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>glass membrane feeders</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>with rubber tubing to fit feeders</td>
</tr>
<tr>
<td>mosquito cups</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>75% ethanol</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10% bleach</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Tissue-culture plates</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>6-well, 24-well and 96-well plates</td>
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<td>Petri dish</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>glass</td>
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<tr>
<td>Slides</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>fine-tip forceps</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1X, sterile</td>
</tr>
<tr>
<td>dissecting microscope</td>
<td>Microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>C6/36 cells</td>
<td>cells</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For virus propagation</td>
</tr>
<tr>
<td>C6/36 medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>MEM with 10% heat inactivated FBS, 1% L-glutamine and non-essential amino acid and 1% (v/v) Penicillin-Streptomycin.</td>
</tr>
<tr>
<td>3&#x2019;-Diaminobenzidine terrahydrochloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D5905</td>
<td>1 tablet in 20 ml of 1 X PBS, dissolve and then add 8 &#181;l of 30% hydrogen peroxidase</td>
</tr>
<tr>
<td>30% hydrogen peroxidase</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>A. aegypti</td>
<td>Animal</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>mosquito</td>
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protocol for dengue infections in mosquitoes (a. aegypti) and infection phenotype determination

この手順の目的は、実験室条件でのデング熱ウイルスにヒトスジシマカの蚊に感染し、蚊の組織の感染レベルやウイルスのダイナミックを検討することである。このプロトコルは、定期的に、特にベクトルの能力を決定することができる新たな宿主因子の同定のために、蚊のウイルスの相互作用を研究するために使用されます。全体の実験は、BSL2実験室で行われなければならない。熱帯熱マラリア原虫の感染症と同様に、手袋と白衣を含む適切な服装を常に着用する必要があります。実験後は、ウイルスと接触したすべての材料は通常の洗濯を始める前に、75％エタノールと漂白で処理する必要があります。他のすべての材料は廃棄それらの前にオートクレーブ処理する必要があります。

Watch this Scientific Journal Video about Protocol for Dengue Infections in Mosquitoes A. aegypti and Infection Phenotype Determination at JoVE.com

Protocol for Dengue Infections in Mosquitoes A. aegypti and Infection Phenotype Determination

遺伝子は、デング熱ウイルスのための潜在的に耐火として識別されると、それは蚊の中でウイルス感染の予防にその役割を評価する必要があります。このプロトコルは、蚊のデング熱感染症の程度をアッセイする方法を示します。文化、膜の給餌蚊、ヒト血液中にウイルスが育ち、そして蚊の中腸におけるウイルス力価を測定するための技術が実証されています。

デング熱蚊の感染症（A.ネッタイシマカ）と感染症の表現型決定のためのプロトコル

The purpose of this procedure is to infect the Aedes mosquito with dengue virus in a laboratory condition and examine the infection level and dynamic of ...

protocol-for-dengue-infections-mosquitoes-aegypti-infection-phenotype

Research

JoVE Journal

Biology

Protocol for Dengue Infections in Mosquitoes (A. aegypti) and Infection Phenotype Determination

17.4K ビュー.  Johns Hopkins University. 遺伝子は、デング熱ウイルスのための潜在的に耐火として識別されると、それは蚊の中でウイルス感染の予防にその役割を評価する必要があります。このプロトコルは、蚊のデング熱感染症の程度をアッセイする方法を示します。文化、膜の給餌蚊、ヒト血液中にウイルスが育ち、そして蚊の中腸におけるウイルス力価を測定するための技術が実証され?...

ビデオ: Protocol for Dengue Infections in Mosquitoes A. aegypti and Infection Phenotype Determination

Microinjection of A. aegypti Embryos to Obtain Transgenic Mosquitoes

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever.  The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever. The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

トランスジェニック蚊を取得するA.ネッタイシマカの胚のマイクロインジェクション

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue ...

生物学

Protocol for Plasmodium falciparum Infections in Mosquitoes and Infection Phenotype Determination

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the mosquito.    This protocol illustrates how the extent of plasmodium infections of mosquitoes can be assayed.  The techniques for preparing the gametocyte culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the mosquito. This protocol illustrates how the extent of plasmodium infections of mosquitoes can be assayed. The techniques for preparing the gametocyte culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

蚊と感染症の表現型決定における熱帯熱マラリア原虫の感染症のためのプロトコル

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the ...

Dissection of Midgut and Salivary Glands from Ae. aegypti Mosquitoes

The mosquito midgut and salivary glands are key entry and exit points for pathogens such as Plasmodium parasites and Dengue viruses. This video protocol demonstrates dissection techniques for removal of the midgut and salivary glands from Aedes aegypti mosquitoes. 



The mosquito midgut and salivary glands are key entry and exit points for vector pathogens like Plasmodium falciparum and the dengue virus. This video demonstrates the dissection techniques for removing the midgut and salivary glands from Aedes aegypti mosquitoes.

AEから中腸と唾液腺の解剖。ネッタイシマカ蚊

The mosquito midgut and salivary glands are key entry and exit points for pathogens such as Plasmodium parasites and Dengue viruses. This video protocol ...

Protocol for RNAi Assays in Adult Mosquitoes (A. gambiae)

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite.  The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Protocol for RNAi Assays in Adult Mosquitoes A. gambiae

Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite. The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

成虫（A. gambiae）におけるRNAiのアッセイのためのプロトコル

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video ...

Preventing the Spread of Malaria and Dengue Fever Using Genetically Modified Mosquitoes

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission.   Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced, as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed.

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission. Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed.

遺伝子組換え蚊を使用してマラリアやデング熱の拡大防止

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission.   Population ...

Building a Better Mosquito: Identifying the Genes Enabling Malaria and Dengue Fever Resistance in A. gambiae and A. aegypti Mosquitoes

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus infections.   Explanation is given for how key refractory genes, those genes conferring resistance to vector pathogens, are identified in the mosquito and how this knowledge can be used to generate transgenic mosquitoes that are unable to carry the malaria parasite or dengue virus.

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus infections. Explanation is given for how key refractory genes, those genes conferring resistance to vector pathogens, are identified in the mosquito and how this knowledge can be used to generate transgenic mosquitoes that are unable to carry the malaria parasite or dengue virus.

より良い蚊の構築：A. gambiaeとA.ネッタイシマカ蚊のマラリアとデング熱の抵抗を使用可能にする遺伝子を特定

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus ...

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. 
This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis1. 
The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

In this protocol we combine RNAi-mediated gene silencing with an in vivo diuresis assay to study the effects knockdown of genes of interest has on mosquito fluid excretion.

RNAiによる遺伝子ノックダウンと<emインビボで&gt;</em大人の女性の&gt;利尿アッセイ<em&gt;ネッタイシマカ</em&gt;蚊

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion ...

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

初代腸オルガノイド培養におけるレトロウイルス感染のビデオプロトコル

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, chikungunya and Zika. Wolbachia-infected mosquitoes can be released into the field to either reduce population sizes through incompatible matings or to transform populations with mosquitoes that are refractory to virus transmission. For these strategies to succeed, the mosquitoes released into the field from the laboratory must be competitive with native mosquitoes. However, maintaining mosquitoes in the laboratory can result in inbreeding, genetic drift and laboratory adaptation which can reduce their fitness in the field and may confound the results of experiments. To test the suitability of different Wolbachia infections for deployment in the field, it is necessary to maintain mosquitoes in a controlled laboratory environment across multiple generations. We describe a simple protocol for maintaining Ae. aegypti mosquitoes in the laboratory, which is suitable for both Wolbachia-infected and wild-type mosquitoes. The methods minimize laboratory adaptation and implement outcrossing to increase the relevance of experiments to field mosquitoes. Additionally, colonies are maintained under optimal conditions to maximize their fitness for open field releases.

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes infected with Wolbachia are being released into natural populations to suppress the transmission of arboviruses. We describe methods to rear Ae. aegypti with Wolbachia infections in the laboratory for experiments and field release, taking precautions to minimize laboratory adaptation and selection.

ネッタイシマカ蚊ボルバキアに感染維持

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

蚊を殺虫化合物小説の毒性試験のプロトコル

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...