蚊におけるRNAiによる遺伝子サイレンシングは、目的の遺伝子を標的と特定のdsRNAの事前準備と浄化が必要です。 dsRNAの合成のため、我々は、in vitro転写反応での T7 RNAポリメラーゼを介したを利用するAmbion社のMegascriptキットをお勧めします。 dsRNAの精製のために、我々は、キアゲンをRNeasyキットをお勧めします。 dsRNAのサンプルを定量化して、水の3μg/μLのために調整する必要があります。あなたが必要とする他の材料は、次のとおり先の細いピンセット、スライドガラス、ガラスマイクロキャピラリー針、マイクロインジェクター（我々はドラモンドのNanoject IIを使用する）、コールドブロック、カバーガラスのペトリ皿、紙タオルや氷のバケツの上にマウントされている光学顕微鏡を。また、10％ショ糖に浸したコットンボールのような食料源と一緒に蚊とそれらに適したコンテナを収集する方法が必要になります。 <ol><li> あなたの蚊を集める 。バッテリ駆動アスピレーターを使用して、注入する蚊を集める。ペーパータオルや綿は蚊がリザーバーをエスケープしないと削除することができるように、ノズルを差し込みます。寒い温度が蚊を麻酔するので、氷のバケツに貯水池を埋めると移動を止めるために蚊に5〜10分待ちます。麻酔は、低温室や冷蔵庫に貯水池を配置することにより、またはCO 2を使用することによって達成することができます。これらの5〜10分時には、次の手順に進みます。 </li><li> あなたの作業領域を準備します 。2 ° Cに平らな面コールドブロックを設定し、その上にスライドガラスを配置。これは、あなたがそれらを注入すると蚊が休息する場所です。インジェクターにガラス針を添付し、目的の幅に針の先端を破るために、ピンセットを使用してください。それは幅が液体のための十分な余地が合理的な速度で流れるように十分な大きさであることが重要であるが、注入中に最小限の傷を負わせるのに十分狭いです。良い針の先端はわずかに曲げるのに十分な狭いかもしれませんが中断されません。希望の出力ボリューム（RNAiに、我々は69 NLを使用）にして噴射装置を設定します。水没してサンプルにして針の先端とメーカーの指示を使用して針にして液体試料を描く。液体サンプルは、dsRNAのソリューション、細菌懸濁液、または他のことができ、注入プロセスはすべて同じです。皿の底が完全に氷に接触されるように氷のバケツにガラスのシャーレを埋め込みます。これは、それらを注入する準備が整うまでは、蚊を置く場所です。 </li><li> あなたの蚊を準備します 。貯水池からのガラスシャーレに50から100蚊を転送。最も彼らは麻酔を維持するために皿の冷たい底部との接触を持っていることを確認して、単層にそれらを薄くするために、ピンセットを使用してください。使用しない時はカバー皿で、これらの蚊をカバーする。蚊の残りの部分に麻酔を保つために氷のバケツに貯水池を返します。 </li><li> コールドブロックに蚊をマウントします 。皿から光顕微鏡下でコールドブロックにマウントされているガラスのスライドに一度に蚊つを転送するように先の細いピンセットを使用してください。望ましいの足で、軽く蚊を扱う。ラインアップスライド上で、多くの30のような蚊のside - by - sideを、あなたがインジェクターとし、それぞれが冷たいスライドに接触することを確認し、それぞれに得ることができることを確認して。あなたの蚊があなたに沿って行くとあなたが持っていると注入されていないものを知るように整然と並んでいることを確認してください。チルドペトリ皿で​​覆われた他の蚊にしておきます。 </li><li> 蚊のあなたの最初のグループを注入する 。片手であなたの先の細い先端がピンセットを持って、鉛筆のような一方で、インジェクターを保持する。あなたが必要として、蚊を移動するには（怪我を避けるために足でそれらを処理する）と、注入のように蚊を安定に保つための鉗子を使用してください。あなたが他の側から注入すると蚊の片側をサポートするための鉗子を使用する場合に最適です。慎重に蚊の胸部に単に針の先端を挿入します。怪我を最小限に抑えるためにキューティクルのピンと張ったまだ薄い部分に注入するのが最善です。また、あまりにも深く注入することは避け、あなただけの先端が胸部の内側になりたい、より深い注射は、生存を危うくされる大きな傷を起こす。針の先端が所定の位置になると、dsRNAのあなたのあらかじめ設定されたボリュームを配信するインジェクター装置のフットペダルを押して放します。 1〜2秒待ってから、蚊から針を取り除く。液体のドロップは、創傷部位の外側に表示された場合、それは傷口に吸収されるまで数秒待ちます。それが数秒以内に吸収されない場合は、その蚊を破棄して再試行してください。あなたのスライドガラス上のすべての蚊の注入工程を継続する。 </li><li> あなたの蚊を格納する 。作業が完了したら、表示した容器内部に注入された蚊を置く（私達がによってカバー1パイントワックス裏地段ボールのカップを好む）ネッティングとラバーバンドと段ボールの蓋で固定メッシュ。 </li><li> 終了するまで注射を続けます 。、スライド上に蚊を配置するために継続注入し、あなたが望むサンプルサイズになるまでカップに移す。一部の死亡 - 初心者のためのアカウントは、多くの蚊は、注射のトラウマから死ぬ必要があります。あなたの蚊のほぼ100％が注射を乗り切るまでは、これで練習して上達するでしょう。 </li><li>終了したら、食の源と一緒に飲みながら折ら、ぬれたペーパータオルを配置し、そのようなプラスチックのラップまたはプラスチックシャーレの蓋などの水分を保持するために何かでカップをカバー。注入された蚊は、ぬれたタオルで提供されるとプラスチックが保持する水分は、これを防ぐに役立つように乾燥しがちです。 </li><li>インキュベーションのための環境制御室で蚊を置きます。 </li></ol> 注 ：遺伝子サイレンシングの効率が非常に多様であり、細胞や臓器によって転写とタンパク質のターンオーバー率、およびdsRNAの取り込み効率を含む多くの要因によって異なります。私たちは、サイレンシングは、1日の注射後には早くも始まる、と6日後に注射するまで持続できることを見出した。定量PCRとウェスタンサイレンシングを検証するためにブロッティングなどの転写産物とタンパク質の検出方法を使用してください。 注 ：蚊注射とRNAiによる遺伝子サイレンシング技術のいくつかのステップでいくつかのバリエーションがあります。いくつかのバリエーションによって記述されています：Boisson、 ら （2006）とブランディンら（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>

遺伝子サイレンシングの効率が大きく変化すると細胞や臓器によって転写とタンパク質のターンオーバー率、およびdsRNAの取り込み効率を含む多くの要因に依存します。私たちは、サイレンシングは、1日の注射後には早くも始まり、6日後に注射するまで持続できることを見出した。定量PCRとウェスタンサイレンシングを検証するためにブロッティングなどの転写産物とタンパク質の検出方法を使用してくだ...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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>
</tr>		</tbody>
		</table>
		</div>

protocol for rnai assays in adult mosquitoes (a. gambiae)

逆遺伝学的アプローチは、蚊のベクトルの病原体にどの遺伝子が基礎となる抵抗を決定するための非常に有用であることが証明されている。このビデオプロトコルは、マラリア原虫を抱いているハマダラカ蚊、にdsRNAを注入するDimopoulosラボで使用される方法を示しています。インジェクションのセットアップを操作し、胸部にdsRNAを注入する手法が示されている。

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 ...

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

Research

JoVE Journal

Biology

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

12.6K ビュー.  Johns Hopkins University. 逆遺伝学的アプローチは、蚊のベクトルの病原体にどの遺伝子が基礎となる抵抗を決定するための非常に有用であることが証明されている。このビデオプロトコルは、マラリア原虫を抱いているハマダラカ蚊、にdsRNAを注入するDimopoulosラボで使用される方法を示しています。インジェクションのセットアップを操作し、胸部にdsRNAを注入する手法が示され...

ビデオ: 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.

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 ...

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.

モスキート子育てのためのプロトコル（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 ...

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 ...

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 ...

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.

哺乳動物細胞におけるRNAiスクリーニングのためのミッション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.

RNAiのエフェクターの細菌配達：Transkingdom RNAiの

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

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

の後胚表現型を識別するためのRNAiスクリーニング C.エレガンス</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

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 ...

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.

キイロショウジョウバエのための持久力トレーニングのプロトコルと縦パフォーマンスアッセイ

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.

マラリア蚊。送信のためのマルチ検出分析

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

Methods Article

成虫（A. gambiae）におけるRNAiのアッセイのためのプロトコル