A.传播病毒在C6/36细胞线。 <ol><li>细胞生长至80％，在75 厘米 2烧瓶汇合; </li><li>删除3-4毫升的容量瓶中剩余的媒体; </li><li>取0.5毫升股票病毒，到上述单元格中添加1.5％细胞的病毒颗粒的多重感染。慢慢地在室温下摇晃15分钟烧瓶。 </li><li> 5％CO 2和37 ° C孵育45分钟</li><li>添加30毫升的媒体，孵育5天。 </li></ol> B.准备病毒和血液的混合物<ol><li>用刮板的细胞分离，细胞和媒体转移到50 ml锥形管; </li><li>离心10分钟;在800克收集上清液，细胞沉淀，但保留上清1毫升; </li><li>冻结上述细胞沉淀在干燥的CO 2，然后在37℃水浴，重复两到三次中解冻; </li><li> 800克为10分钟，离心取上清液，并结合从第3步收集上清; </li><li>收集与编制配子体文化感染与恶性疟原虫的按蚊蚊子相同的步骤（步骤3）整个人类的血液; </li><li>结合上述整个人体血液中病毒从第4步上清的平等，并加入人血清（整个体积的10％）。 </li><li> 30分钟，在37℃水浴孵育上述混合物对人体血液和登革热病毒</li></ol> C.喂养蚊子此过程一直在恶性疟原虫在蚊子感染的部分描述几乎是相同的。在第7天，我们检测肠病毒感染，吸血后第14天的唾液感染。在与病毒接触的所有材料的处理，首先用75％乙醇，然后用10％漂白粉。 D.含量的蚊子组织中的病毒滴度<ol><li>蚊子组织清扫术前两三天，增长C6/36细胞在24孔板的细胞达到80％，在检测时进行一天的汇合; </li><li>蚊子麻醉在4 ° C时，75％的乙醇浸泡1分钟，无菌牺牲浮出水面。蚊子肠或唾液腺解剖解剖显微镜下用无菌水冲洗，前两次。在此步骤中，所有下面的程序需要在无菌的环境，如生物安全柜中进行。这些组织被转移到管内含有150μL媒体和匀浆一个Kontes颗粒杵电机为90秒; </li><li> 10匀浆液加入到990μL媒体1:100稀释; </li><li>使进一步的1：10，1:100和1:1000稀释介质，在96孔板; </li><li>媒体在24孔板中删除，并添加100μL上述四个稀释，每年每个相应的井; </li><li>板块缓慢摇动15分钟，在室温下孵育45分钟，然后在5％，CO 2和37℃; </li><li> methycellulose叠加（1％）加1ml到每口井; </li><li>板在37 ° C的5％的CO 2为5天; </li><li>从这里开始的步骤并不需要无菌的环境，但应在任何时候都采取与任何其他的病原体护理。丢弃从每个板块methycellulose覆盖和污点去除多余媒体</li><li>添加甲醇/丙酮固定液（1:1）1ml到​​每口井。在4 ° C为1小时保持; </li><li>倒掉固定液，用1X PBS清洗一次; </li><li> 1:1000稀释的5％烂醉的对抗病毒的抗体; </li><li> 200μL稀释高免液添加到每个井，37孵育° C为1小时; </li><li>删除高免1 × PBS液和洗板</li><li>准备在5％烂醉的（5G脱脂牛奶100毫升为1 × PBS）1:1000稀释的羊抗鼠共轭（嘉里建设猫＃074-1806），然后每孔加入200μl，孵育37℃1小时; </li><li>准备基板如下：1 3&#39; -二氨基terrahydrochloride片剂（西格玛猫＃D5905），添加20毫升1 × PBS，溶解后，再加入8μL30％的氢过氧化物酶</li><li>每孔加入200μL上述基板。让我们在室温下站10分钟</li><li>取出的基材，并停止加入蒸馏水1ml到每口井的反应</li><li>倒入水和杂交板干</li><li>计数的病毒粒子</li></ol>

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		<div>
		<table border="1">
		<thead>
		<tr>
		<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>
<tr>
<td>Tissue-culture plates</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>6-well, 24-well and 96-well plates</td>
</tr>
<tr>
<td>Petri dish</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>glass</td>
</tr>
<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>
</tr>		</tbody>
		</table>
		</div>

protocol for dengue infections in mosquitoes (a. aegypti) and infection phenotype determination

这一程序的目的是感染登革热病毒的伊蚊，在实验室条件和研究感染病毒在蚊子的组织水平和动态。该协议是经常用于研究蚊子病毒的相互作用，特别是确定新的宿主因素，能够确定载体能力。在整个实验必须在BSL2实验室进行。类似恶性疟原虫感染，在任何时候都必须佩戴适当的服装，包括手套和白大褂。实验结束后，所接触到的病毒的所有材料需要75％的乙醇，然后再进行正常洗涤漂白处理。需要抛弃他们之前蒸压的所有其他材料。

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

一旦一个基因被确定为潜在的顽固性登革热病毒，它必须进行评估，它在防止病毒感染的蚊子作用。该协议说明登革热的蚊子感染的程度如何，可以检测。成长的文化，膜喂养蚊子人体血液中的病毒，并化验在蚊子中肠病毒滴度的技术演示。

在蚊子（埃及斑蚊）感染引起的表型测定为登革热感染病例的议定书“

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 次观看.  Johns Hopkins University. 一旦一个基因被确定为潜在的顽固性登革热病毒，它必须进行评估，它在防止病毒感染的蚊子作用。该协议说明登革热的蚊子感染的程度如何，可以检测。成长的文化，膜喂养蚊子人体血液中的病毒，并化验在蚊子中肠病毒滴度的技术演示。

视频: 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.

埃及斑蚊胚胎显微注射获得转基因蚊子

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

科研

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.

恶性疟原虫在蚊子和病毒感染的表型测定感染的议定书

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.

从AE肠和唾液腺的解剖。埃及伊蚊蚊

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.

成蚊（冈比亚按蚊的RNAi实验议定书）

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.

防止疟疾的传播和使用基因改造的蚊子登革热

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.

建设一个更美好的蚊子：确定基因在冈比亚按蚊和埃及斑蚊蚊子启用疟疾和登革热的电阻

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介导的基因敲除和<em&gt;在体内</em在成年女性利尿含量<em&gt;埃及伊蚊</em&gt;蚊子

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.

逆转录病毒感染的原发性肠化培养的视频协议

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.

维护伊蚊蚊蚊子感染沃尔巴克

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.

新型杀虫化学对蚊子毒性测试规程

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