RNAi介导的基因沉默在蚊子需要事前准备和提纯感兴趣的基因的具体dsRNAs目标。双链RNA的合成，我们建议Ambion公司Megascript套件，这使得使用一个T7 RNA聚合酶介导的体外转录反应。对于纯化的双链RNA，我们建议Qiagen公司的RNeasy试剂​​盒。双链RNA的样品应量化，并调整为3微克/μL在水中。其他材料，您需要包括：细尖镊子，载玻片，玻璃microcapillary针，微量（我们使用Drummond的Nanoject第二），一个光显微镜安装以上冷块，一个涵盖玻璃的Petri菜，纸巾和一桶冰。您还需要一种方法来收集蚊子和适合自己的容器，随着食物来源，如浸泡在10％蔗糖的棉花球。 <ol><li> 收集您的蚊子 。使用电池供电的抽吸，收集你想注入的蚊子。将喷嘴用纸巾或棉花，所以没有蚊子能逃脱和消除水库。由于寒冷的气温麻醉蚊子，埋在冰桶水库和等待5-10分钟停止移动的蚊子。放置在冷室或冰箱水库或使用的CO 2，麻醉也可以实现。在5-10分钟，继续下一步。 </li><li> 准备好您的工作区设置一个平面冷块至2 ° C和地方上的载玻片。这是蚊子的地方休息，你注入。连接到微量的玻璃针和使用您的镊子打破针尖到所需的宽度。重要的是，宽度是大到足以让足够的空间用于液体流在一个合理的速度通过​​，但很窄，足以造成注射过程中的一个最小的伤​​口。一个良好的针头将足够窄，稍微弯曲，但不会被打破。设置您的注射设备所需的输出量（用于RNAi，我们使用69 NL）。淹没尖端进入你的样品针吸引到您的液体样品使用制造商的指示针。液体样品可以dsRNA的解决方案，细菌悬浮液，或其他注射过程是所有相同。在冰桶中嵌入的玻璃培养皿中，使盘底部完全接触冰。这是在那里你会放，直到您准备注入的蚊子。 </li><li> 准备好您的蚊子 。转让50-100蚊子从玻璃培养皿水库。使用钳薄的单层，确保大部分的菜冷底部接触，以保持他们的麻醉。菜盖盖时，在不使用这些蚊子。返回水库冰桶，保持休息的蚊子麻醉。 </li><li> 冷块山蚊子 。使用细尖钳一次传输的蚊子从盘安装在光学显微镜下的冷块的载玻片。把手轻轻的蚊子，最好的腿。多达30个蚊子在幻灯片上侧端线，确保你可以得到每一个与微量，并确保每一个与寒冷的幻灯片接触。确保您的蚊子都排队有序，让你走，你知道有和没有被注入。保留在冷冻的培养皿中涉及的其他蚊子。 </li><li> 注入蚊子的第一组。一方面保持细尖的尖钳，像铅笔另一方面举行的微量。使用镊子移动蚊子，因为你需要的（处理他们，以避免受伤的腿），​​并保持稳定，为你注入蚊子。这最好的作品，如果你使用的镊子支持蚊子的一面，当你从另一个侧面注入。只是针尖小心地放入蚊子的胸部。最好是一个绷紧的角质层，以尽量减少伤害而薄的一部分注入。同时，避免注射过深，你想只是冰山胸廓内，更深的注射造成更大的创伤会妥协的生存。一旦针尖位置，按下并释放微量设备将提供您预先设定的dsRNA量的脚踏板。等待一两秒钟，消​​除蚊子的针。如果液滴出现伤口外，等待几秒钟，它是在伤口吸收。如果它不吸收，在几秒钟内，丢弃，蚊子，然后再试一次。继续为您的载玻片上所有蚊子的注射过程。 </li><li> 商店的蚊子 。当你完成后，将有标签的容器内注入蚊子（我们宁愿1品脱蜡内衬纸板覆盖杯网状扣除，并用橡皮筋和纸板盖担保）。 </li><li> 继续注射，直至完成 。继续把蚊子在幻灯片上，注入和转移到杯子，直到你有你的愿望的样本大小。对于一些初学者死亡的帐户将有许多蚊子死于注射的创伤。这将改善与实践，直到几乎100％的蚊子在生存注射。 </li><li>当完成后，将一起食物来源比杯折叠，湿纸巾和保留水分，如保鲜膜或塑料的Petri盖盖的杯子。注射蚊子很容易干燥，所以提供的湿毛巾和塑料保留的水分会有助于防止这种。 </li><li>放置在孵化室环境控制蚊子。 </li></ol> 注 ：基因沉默的效率是高度可变的，取决于许多因素，包括成绩单和蛋白质的转，超过利率，并通过细胞和器官的双链RNA吸收效率。我们发现，沉默开始早1天注射后，可以持续长达6天注射后。使用成绩单和蛋白质的检测方法，如定量PCR和Western印迹，以验证沉默。 注 ：有几个变化对蚊子的注射和RNAi介导的基因沉默技术的几个步骤。所描述的一些变化：布瓦松，等人（2006年）和Blandin，等（2002） 。

<ol>
	<li>Boisson, ., 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> , (2006).</li><li>Blandin, ., 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> , (2002).</li></ol>

基因沉默的效率是高度可变的，取决于多项因素，包括成绩单和蛋白质的转，超过利率，并通过细胞和器官的双链RNA吸收效率。我们发现，沉默开始早在1天注射后，可长达6天注射后。使用成绩单和蛋白质的检测方法，如定量PCR和Western印迹，以验证沉默。

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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>Megascript</td>
<td>kit</td>
<td>Ambion</td>
<td>&nbsp;</td>
<td>for dsRNA synthesis; T7 RNA polymerase-mediated in vitro transcription reaction</td>
</tr>
<tr>
<td>RNeasy</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>&nbsp;</td>
<td>For dsRNA purification</td>
</tr>
<tr>
<td>dsRNA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>should be quantified and adjusted to 3 &#x03BC;g/ &#x03BC;L in water</td>
</tr>
<tr>
<td>fine-tipped forceps</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>glass slide</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>glass microcapillary needles</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>microinjector</td>
<td>&nbsp;</td>
<td>Drummond</td>
<td>Nanoject II</td>
<td>&nbsp;</td>
<tr>
<td>light microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>mounted above a cold block</td>
</tr>
<tr>
<td>Petri dish</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>covered, glass</td>
</tr>
<tr>
<td>paper towels</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10% sucrose</td>
<td>food</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>A. gambiae</td>
<td>Animal</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>mosquitos</td>
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protocol for rnai assays in adult mosquitoes (a. gambiae)

反向遗传方法已被证明非常有用，确定哪些基因底层电阻载体病原体在蚊子。该视频协议说明Dimopoulos实验室使用的冈比亚按蚊的蚊子，其中海港的疟疾寄生虫注入dsRNA的一个方法。操纵注射的设置和注入胸廓的dsRNA的技术说明。

Watch this Scientific Journal Video about Protocol for RNAi Assays in Adult Mosquitoes A. gambiae at JoVE.com

Protocol for RNAi Assays in Adult Mosquitoes A. gambiae

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 ...

protocol-for-rnai-assays-in-adult-mosquitoes-a-gambiae

Research

JoVE Journal

Biology

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

12.6K 次观看.  Johns Hopkins University. 反向遗传方法已被证明非常有用，确定哪些基因底层电阻载体病原体在蚊子。该视频协议说明Dimopoulos实验室使用的冈比亚按蚊的蚊子，其中海港的疟疾寄生虫注入dsRNA的一个方法。操纵注射的设置和注入胸廓的dsRNA的技术说明。

视频: Protocol for RNAi Assays in Adult Mosquitoes A. gambiae

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

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 the virus in the mosquito tissues. This protocol is routinely used for studying mosquito-virus interactions, especially for identification of novel host factors that are able to determine vector competence. The entire experiment must be conducted in a BSL2 laboratory. Similar to Plasmodium falciparum infections, proper attire including gloves and lab coat must be worn at all times. After the experiment, all the materials that came in contact with the virus need to be treated with 75% ethanol and bleached before proceeding with normal washing. All other materials need to be autoclaved before discarding them.

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

Once a gene is identified as potentially refractory for the dengue virus, it must be evaluated for it's role in preventing viral infections within the mosquito. This protocol illustrates how the extent of dengue infections of mosquitoes can be assayed. The techniques for growing up the virus in culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

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 ...

科研

生物学

Protocol for Mosquito Rearing (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night cycle. For this procedure, you'll need mosquito cages, 10% sterile sucrose solution, paper towels, beaker, whatman filter paper, glass feeders, human blood and serum, water bath, parafilm, distilled water, clean plastic trays, mosquito food (described below), mosquito net to cover the trays, vacuum, and a collection chamber to collect adults.

Protocol for Mosquito Rearing A. gambiae

This video illustrates the general techniques used to rear Anopheles gambiae in the laboratory. The methods for caring for laboratory mosquitoes are demonstrated through all stages of the organism's life cycle from larvae to pupae to blood-feeding adults.

蚊子养育的议定书“（冈比亚按蚊）

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night ...

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 ...

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 ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

在哺乳动物细胞的RNAi筛选使命esiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory tool, the RNAi pathway appears to be promising for therapeutic utilization. For development of RNA interference (RNAi)-based therapies, delivery of RNAi-mediating agents to target cells is one of the major obstacles. A novel strategy to overcome this hurdle is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria, e.g. Escherichia coli, to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. A first-generation tkRNAi-mediating vector, TRIP, contains the bacteriophage T7 promoter for expression regulation of a therapeutic shRNA of interest. Furthermore, TRIP has the Inv locus from Yersinia pseudotuberculosis that encodes invasin, which permits natural noninvasive bacteria to enter &beta;1-integrin-positive mammalian cells and the HlyA gene from Listeria monocytogenes, which produces listeriolysin O. This enzyme allows the therapeutic shRNA to escape from entry vesicles within the cytoplasm of the target cell. TRIP constructs are introduced into a competent non-pathogenic Escherichia coli strain, which encodes T7 RNA polymerase necessary for the T7 promoter-driven synthesis of shRNAs. A well-characterized cancer-associated target molecule for different RNAi strategies is ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). This ABC-transporter acts as a drug extrusion pump and mediates the "classical" ABCB1-mediated multidrug resistance (MDR) phenotype of human cancer cells which is characterized by a specific cross resistance pattern. Different ABCB1-expressing MDR cancer cells were treated with anti-ABCB1 shRNA expression vector bearing E. coli. This procedure resulted in activation of the RNAi pathways within the cancer cells and a considerable down regulation of the ABCB1 encoding mRNA as well as the corresponding drug extrusion pump. Accordingly, drug accumulation was enhanced in the pristine drug-resistant cancer cells and the MDR phenotype was reversed. By means of this model the data provide the proof-of-concept that tkRNAi is suitable for modulation of cancer-associated factors, e.g. ABCB1, in human cancer cells.

For development of RNA interference (RNAi)-based therapies, a novel strategy was developed, transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells. Here, tkRNAi was successfully applied for reversal of classical ABCB1-mediated multidrug resistance (MDR) of cancer cells.

细菌提供的RNAi效应：Transkingdom RNA干扰

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory ...

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 ...

Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster

One of the most pressing problems facing modern medical researchers is the surging levels of obesity, with the consequent increase in associated disorders such as diabetes and cardiovascular disease 1-3. An important topic of research into these associated health problems involves the role of endurance exercise as a beneficial intervention.
Exercise training is an inexpensive, non-invasive intervention with several beneficial results, including reduction in excess body fat 4, increased insulin sensitivity in skeletal muscle 5, increased anti-inflammatory and antioxidative responses 6, and improved contractile capacity in cardiomyocytes 7. Low intensity exercise is known to increase mitochondrial activity and biogenesis in humans 8 and mice, with the transcriptional coactivator PGC1-&alpha; as an important intermediate 9,10. 
Despite the importance of exercise as a tool for combating several important age-related diseases, extensive longitudinal genetic studies have been impeded by the lack of an endurance training protocol for a short-lived genetic model species. The variety of genetic tools available for use with Drosophila, together with its short lifespan and inexpensive maintenance, make it an appealing model for further study of these genetic mechanisms. With this in mind we have developed a novel apparatus, known as the Power Tower, for large scale exercise-training in Drosophila melanogaster 11. The Power Tower utilizes the flies' instinctive negative geotaxis behavior to repetitively induce rapid climbing. Each time the machine lifts, then drops, the platform of flies, the flies are induced to climb. Flies continue to respond as long as the machine is in operation or until they become too fatigued to respond. Thus, the researcher can use this machine to provide simultaneous training to large numbers of age-matched and genetically identical flies. Additionally, we describe associated assays useful to track longitudinal progress of fly cohorts during training.

Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster

We describe the first endurance training protocol for an important genetic model species, Drosophila melanogaster, and outline several assays to chart improvements in mobility following training.

果蝇的耐力训练协议和纵向性能检测

One of the most pressing problems facing modern medical researchers is the surging levels of obesity, with the consequent increase in associated disorders ...

A Multi-detection Assay for Malaria Transmitting Mosquitoes

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, several traits are typically characterized using molecular assays to aid in epidemiological studies. These traits include species identification, insecticide resistance, parasite infection status, and host preference. Since populations of the Anopheles gambiae complex are morphologically indistinguishable, a polymerase chain reaction (PCR) is traditionally used to identify species. Once the species is known, several downstream assays are routinely performed to elucidate further characteristics. For instance, mutations known as KDR in a para gene confer resistance against DDT and pyrethroid insecticides. Additionally, enzyme-linked immunosorbent assays (ELISAs) or Plasmodium parasite DNA detection PCR assays are used to detect parasites present in mosquito tissues. Lastly, a combination of PCR and restriction enzyme digests can be used to elucidate host preference (e.g., human vs. animal blood) by screening the mosquito bloodmeal for host-specific DNA. We have developed a multi-detection assay (MDA) that combines all of the aforementioned assays into a single multiplex reaction genotyping 33SNPs for 96 or 384 samples at a time. Because the MDA includes multiple markers for species, Plasmodium detection, and host blood identification, the likelihood of generating false positives or negatives is greatly reduced from previous assays that include only one marker per trait. This robust and simple assay can detect these key mosquito traits cost-effectively and in a fraction of the time of existing assays.

Malaria transmitting mosquitoes have a number of epidemiologically important characteristics that can only be detected using molecular techniques. Utilizing a MALDI-TOF based SNP genotyping platform, we developed an assay for simultaneously detecting multiple key traits (species, insecticide resistance, parasite infection and host choice) of malaria vectors.

一种多检测试验为疟疾蚊子发射

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, ...

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory for functional genomic studies. The ability to knockdown individual genes selectively via this reverse genetic technique has allowed many researchers to rapidly uncover the biological roles of numerous genes within many organisms, by evaluation of loss-of-function phenotypes. In the major human malaria vector Anopheles gambiae, the predominant method used to reduce the function of targeted genes involves injection of double-stranded (dsRNA) into the hemocoel of the adult mosquito. While this method has been successful, gene knockdown in adults excludes the functional assessment of genes that are expressed and potentially play roles during pre-adult stages, as well as genes that are expressed in limited numbers of cells in adult mosquitoes. We describe a method for the injection of Serine Protease Inhibitor 2 (SRPN2) dsRNA during the early pupal stage and validate SRPN2 protein knockdown by observing decreased target protein levels and the formation of melanotic pseudo-tumors in SRPN2 knockdown adult mosquitoes. This evident phenotype has been described previously for adult stage knockdown of SRPN2 function, and we have recapitulated this adult phenotype by SRPN2 knockdown initiated during pupal development. When used in conjunction with a dye-labeled dsRNA solution, this technique enables easy visualization by simple light microscopy of injection quality and distribution of dsRNA in the hemocoel.

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi) is an extremely valuable tool for uncovering gene function. However, the ability to target genes using RNAi during pre-adult stages is limited in the major human malaria vector Anopheles gambiae. We describe an RNAi protocol to reduce gene function via direct injection during pupal development.

RNAi的触发传送到<em&gt;冈比亚按蚊</em&gt;蛹

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory ...