A.이 C6/36 세포주에 바이러스를 전파. <ol><li> 세포는 75cm이 플라스크에 confluency 80 % 성장; </li><li> 술병에 남아 3-4 ML과 미디어를 제거; </li><li> 0.5 ML 주식 바이러스를 가지고 세포 당 1.5 바이러스 입자의 감염의 다중성과 함께 위의 셀에 추가합니다. 서서히 실온에서 15 분 동안 휴대용 술병을 흔들어. </li><li> 5% CO 2와 37 ° C에서 45 분 동안 품어 </li><li> 30 ML 미디어를 추가, 5 일 동안 알을 품다. </li></ol> B. 바이러스와 혈액의 혼합물 준비 <ol><li> 스크레이퍼로 세포를 분리하고 50 ML 원뿔 튜브에 세포와 미디어를 전송; </li><li> 10 분 800g에 Centrifugate, 표면에 뜨는를 수집하지만, 세포 펠렛과 함께 뜨는의 1ml를 떠나; </li><li> ° C 물 목욕, 반복 2 ~ 3 배 37 해동 후 건조 CO 2에 위의 세포 펠렛을 고정하고, </li><li> 뜨는을하고, 3 단계에서 수집한 뜨는와 결합, 10 분 800g에 Centrifugate; </li><li> Plasmodium falciparum과 아노 펠 레스 모기를 감염시킬 수 gametocyte 문화의 준비에 대해 동일한 절차 (3 단계)와 전체 인간의 혈액을 수집; </li><li> 4 단계에서 바이러스 뜨는와 함께 위의 모든 인간의 혈액의 동일한 금액을 결합, 그리고 (전체 볼륨의 10 %) 인간의 혈청을 추가합니다. </li><li> 37 ° C 물 목욕 30 분 인간의 혈액과 뎅그열 바이러스의 상기 혼합물을 품어 </li></ol> C. 먹이 모기 이 절차는 모기에 Plasmodium falciparum 감염에 대한 섹션에서 설명했던 일들을 거의 동일합니다. 우리는 일 7시 분석 midgut 감염, 피로 수유 후 14 일에서 타액 감염. 바이러스와 접촉에 온 모든 자료는 75 % 에탄올과 10 %의 다음 표백제 처음에 처리됩니다. D. 어세 모기 조직에서 바이러스 titer <ol><li> 2-3 일 모기 조직의 절개 전에 세포는 분석이 실시되는 하루 confluency 80 %에 도달 이러한 24 잘 접시에 C6/36 세포를 성장; </li><li> 모기가 4 anesthetized 아르 ° C, 희생과 1 분 75 % 에탄올에서 그들을 담글하여 멸균 표면. 그렇다면 모기는 해부 현미경 midgut 또는 침샘의 해부 전에 두 번 멸균 물로 씻어되었습니다. 이 단계에서 아래의 모든 절차는 생물 학적 안전 캐비닛 같은 무균 환경에서 실시해야합니다. 이러한 조직은 150 μl 미디어를 포함하는 튜브에 옮겨 90 초간 Kontes 펠렛 유봉 모터 균질되며 </li><li> 990 μl 미디어에 homogenate 10 μl를 추가하여 1:100 희석하다; </li><li> 추가 1 확인 : 10, 1:100 및 96 잘 판에 매체와 1:1000 희석을; </li><li> 24 잘 판에있는 미디어를 제거하고 각 해당 잘하려면 위의 네 dilutions 각각 100 μl를 추가합니다; </li><li> 그리고 5 %에서 45 분 동안 부화 상온에서 15 분에 대해 천천히 접시를 흔들어 CO 2 37 ° C; </li><li> 각 잘하는 프로필 메틸 셀룰로오스 오버레이 (1 %)의 1ml을 추가합니다; </li><li> 37 접시를 품어 ° C 오일에 대한 5 % CO 2; </li><li> 여기에서 단계 멸균 환경을 필요로하지 않지만 같은 다른 병원체주의는 항상 주의해야한다. 각 플레이트의 프로필 메틸 셀룰로오스 오버레이를 취소하고 여분 미디어를 제거 얼룩 </li><li> 각 잘하는 메탄올 / 아세톤 정착액 (1:1)의 1ml을 추가합니다. 1 시간 4 ° C에 보관; </li><li> 정착액을 붓고하고, 1X PBS로 한번 씻어; </li><li> 5% 곤드레 만 드레 취한와 바이러스에 대한 기본 항체에 1:1000 희석하다; </li><li> 1 시간 각 37 품어, 잘 ° C로 희석 hyperimmune 액체 200 μl를 추가합니다; </li><li> 1 X PBS로 hyperimmune 유체 및 세척 플레이트를 제거합니다 </li><li> 5% 곤드레 만 드레 취한 (1 X PBS의 100 ML에 5g 분말 탈지 우유)에 염소 안티 마우스 공액의 1:1000 희석 (KPL 캣 # 074-1806)를 준비하고, 다음 각 잘 200 μl을 추가하고에서 알을 품다 1 시간을위한 37 ° C; </li><li> 다음과 같이 기판을 준비 : 해산 후, 1 X PBS의 20 ML에 3&#39; - Diaminobenzidine terrahydrochloride 1 타블렛 (시그마 고양이 # D5905)을 추가하고 다음 30 % 수소 퍼옥시데이즈 8 μl를 추가 </li><li> 각 잘하려면 위의 기판 200 μl를 추가합니다. 10 분 실온에서 서합시다 </li><li> 기판을 제거하고 각 잘하는 증류수의 1ml을 추가하여 반응을 정지 </li><li> 건조에 물 얼룩 접시를 부어 </li><li> 바이러스 입자를 카운트 </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

이 절차의 목적은 실험실 조건에서 뎅그열 바이러스 Aedes의 모기를 감염하고 모기가 조직의 감염 수준 및 바이러스의 역학을 조사하는 것입니다. 이 프로토콜은 정기적으로, 특히 벡터 능력을 확인할 수있는 새로운 호스트 요인의 식별, 모기 바이러스 상호 작용을 연구하는 데 사용됩니다. 전체 실험은 BSL2의 연구실에서 수행되어야합니다. Plasmodium falciparum 감염과 마찬가지로, 장갑과 실험 복을 포함한 적절한 복장은 항상 착용해야합니다. 실험 후, 바이러스와 접촉 온 모든 자료는 일반 세탁기와 함께 진행하기 전에 75 % 에탄올과 표백로 치료해야합니다. 다른 모든 자료는 폐기 앞에 autoclaved해야합니다.

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

유전자가 뎅그열 바이러스에 대한 잠재적 내화물로 식별되면, 그것은 모기 내에서 바이러스 감염을 방지에의 역할에 대해 평가해야합니다. 이 프로토콜은 모기의 뎅그열 감염의 범위가 assayed 수있는 방법을 보여줍니다. 문화, 멤브레인 수유 모기 인간의 혈액에있는 바이러스를 성장하고, 모기 midgut에서 바이러스 titers을 시금에 대한 기술은 증명됩니다.

모기 (A. aegypti)와 감염 표현형 결정에 뎅그열 감염 프로토콜

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 ...

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

17.4K 조회수.  Johns Hopkins University. 유전자가 뎅그열 바이러스에 대한 잠재적 내화물로 식별되면, 그것은 모기 내에서 바이러스 감염을 방지에의 역할에 대해 평가해야합니다. 이 프로토콜은 모기의 뎅그열 감염의 범위가 assayed 수있는 방법을 보여줍니다. 문화, 멤브레인 수유 모기 인간의 혈액에있는 바이러스를 성장하고, 모기 midgut에서 바이러스 titers을 시금에 대한 기술은 증명됩니다.

비디오: 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.

트렌스 제닉 모기를 구하는 A. aegypti의 태아의 Microinjection

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

연구

생물학

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.

모기와 감염 표현형 결정에서 Plasmodium falciparum 감염에 대한 프로토콜

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.

애에서 Midgut과 침샘의 해부. aegypti 모기

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.

성인 모기 (A. gambiae)에서 RNAi의 Assays을위한 프로토콜

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.

유전자 변형 모기를 사용하여 말라리아와 뎅그열 피버의 확산을 방지

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.

더 나은 모스키토 구축 : A. gambiae 및 A. aegypti 모기에 말라리아와 뎅그열 발열 저항을 사용 유전자를 확인

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 - 중재 유전자 최저 및<em&gt; VIVO에서</em성인 여자의&gt; 이뇨 분석<em&gt; Aedes aegypti</em&gt; 모기

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.

차 소장 Organoid 문화에서 레트로 바이러스 감염의 비디오 프로토콜

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.

Aedes aegypti 모기 감염 Wolbachia 유지

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.

모기에 소설 Insecticidal 화학 물질의 독성을 테스트 하기 위한 프로토콜

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