모기에 RNAi - 중재 유전자 입을 관심의 유전자를 대상으로 특정 dsRNAs의 사전 준비 및 정화가 필요합니다. dsRNA 합성을 위해, 우리는 T7 RNA 시험 관내 전사 반응에서 효소 - 중재의 사용을 만드는 앰비온의 Megascript 키트를 권장합니다. dsRNA 정화를위한, 우리는 Qiagen RNeasy 키트를 권장합니다. dsRNA의 샘플은 계량 3 μg / 물 μL 조정해야합니다. 당신이 필요합니다 다른 자료는 다음과 같습니다 미세 밀고 포셉, 유리 슬라이드, 유리 microcapillary 바늘, microinjector (우리가 드루먼드의 Nanoject II를 사용하여), 감기에 블록, 커버 유리 페트리 접시, 종이 타월과 얼음 양동이 위에 장착 가벼운 현미경 . 당신은 또한 10 %의 자당에 배어 목화 공을 같은 음식 소스와 함께 모기 그들에 적합한 컨테이​​너를 수집하는 방법이 필요합니다. <ol><li> 당신의 모기를 수집합니다. 배터리 구동 흡인기를 사용하면 삽입하고자하는 모기를 수집합니다. 종이 타월이나 면으로도 모기가 저수지 탈출하지 않고 제거할 수있는 노즐​​을 꽂습니다. 추운 기온이 모기를 마취 때문에, 얼음 양동이에있는 저수지 매장 및 이동 막기 위해 모기에 대한 50-10분을 기다립니다. Anesthetization도 감기 방이나 냉장고에 저수지를 삽입하거나 CO 2를 사용하여 얻을 수 있습니다. 이러한 5-10분 동안 다음 단계를 계속합니다. </li><li> 작업 영역을 준비합니다. 2 평평한 표면 감기에 블록을 설정 ° C와 거기에 유리 슬라이드를 놓으십시오.이 이것은 당신이 주사로 모기가 나머지됩니다 곳입니다. microinjector에 유리 바늘을 첨부하고 원하는 너비로 바늘의 팁을 휴식에 집게를 사용합니다. 넓이가 액체에 대한 충분한 공간이 적당한 속도로 흐르는 수 있도록 충분히 큰지만 주입하는 동안 최소한의 상처를 입힐 수있을만큼 좁고 것이 중요합니다. 좋은 바늘 끝이 약간 구부 정도로 좁은 수 있지만 휴식하지 않습니다. 원하는 출력 볼륨 (RNAi을 위해, 우리는 69 NL을 사용)로 분사 장비를 설정합니다. 잠수함은 샘플에 바늘의 팁 및 제조 업체의 지침을 사용하여 바늘에 액체 샘플을 그립니다. 액체 시료는 dsRNA 솔루션 세균 정지 또는 기타 수 있으며, 사출 과정은 모두에 대해 동일합니다. 접시의 바닥이 완전히 얼음을 연락되도록 얼음 양동이에 유리 페트리 접시를 삽입할 수 있습니다. 당신이 그들을 주입 준비가 될 때까지 당신은 모기를 놓습니다 곳입니다. </li><li> 당신의 모기를 준비합니다. 전송 50-100 모기 유리 페트리 접시에 저수지에서. 대부분의 그들이 anesthetized 유지하는 요리의 차가운 바닥과 접촉했는지 확인하고, 단일 레이어로 얇은 위해 집게를 사용하십시오. 사용하지 않을 때는 커버 접시 이러한 모기 커버. 모기의 나머지 anesthetized 유지하는 얼음 양동이에 저수지를 반환합니다. </li><li> 추운 블록에 모기를 탑재합니다. 접시에서 빛을 현미경 추위 블록에 장착된 유리 슬라이드에 한 번에 모기를 전송하여 미세 밀고 집게를 사용하십시오. 낫게 다리에 의해 부드럽게 모기를 처리할 수 있습니다. 서 슬라이드 많은 30 모기 나란히, 당신이 microinjector 각 하나를 얻을 각 하나가 감기 슬라이드와 접촉하게 확인하고 확신하고. 당신의 모기가 당신이 따라가는대로하고 주입되지 않은 어떤 알 수 있도록 질서 줄지어 있는지 확인합니다. 냉장 페트리 접시에 나와 다른 모기를 남겨주세요. </li><li> 모기의 첫 번째 그룹을 주사. 한 손으로에서 미세 스쳐 스쳐 집게를 잡고 연필처럼 한편의 microinjector를 개최. 필요로 모기를 이동 (부상을 피하기 위해 다리하여 처리) 및 주사로 모기가 안정 유지하기 포셉을 사용합니다. 당신은 다른 측면에서 주사로이 모기의 한쪽을 지원하는 포셉을 사용하는 경우가 최상의 상태로 작동합니다. 조심스럽게 모기의 흉부에 그냥 바늘 끝부분을 삽입합니다. 부상을 최소화하기 위해 표피의 긴장 늦추지 아직 얇은 부분에 주입하는 것이 좋습니다. 또한, 너무 깊이 주입하지 않도록, 당신은 흉부 내부로 끝에만 원하는, 깊은 주사는 생존을 위해서야 큰 상처의 원인이됩니다. 일단 바늘 끝이 위치에, 언론과 dsRNA의 사전 설정된 볼륨을 제공합니다 microinjector 장치의 발을 페달을 놓습니다. 두 번째 또는 두 기다려하고 모기에서 바늘을 제거합니다. 액체 한 방울의 상처 사이트 외부 나타나면 그것이 상처로 흡수 될 수있는 몇 초 정도 기다리십시오. 그것은 몇 초 내에 흡수하지 않는 경우, 그 모기를 버리고 다시 시도하십시오. 당신의 유리 슬라이드에있는 모든 모기에 대한 사출 과정을 계속합니다. </li><li> 당신의 모기를 저장합니다. 작업이 완료되면 표시 용기 안에 주입 모기 플레이스 (우리는이 적용 한 파인트 왁스 늘어선 종이 컵을 선호) 그물 및 고무 밴드 및 판지 뚜껑과 보안을 메쉬. </li><li> 완료까지 주사를 계속합니다., 슬라이드에 모기를 게재할 계속 주사하고 여러분이 원하는 샘플 크기를 때까지 컵을 전송할 수 있습니다. 어떤 죽음 - 초보자를위한 계정은 많은 모기가 주사의 외상에서 죽게됩니다. 당신의 모기의 거의 100 %가 주사를 살아남을 때까지이 연습 향상됩니다. </li><li> 완료되면, 음식 소스와 함께 한잔으로 접으면, 젖은 종이 타월을 장소와 같은 플라스틱 랩 또는 플라스틱 페트리 뚜껑으로 수분을 유지하기 위해 뭔가 컵을 커버. 주입 모기는 젖은 수건으로 제공하고 플라스틱으로 유지 수분이되지 않도록 도움이 될 것입니다 그러니 건조하는 경향이 있습니다. </li><li> 배양을위한 환경 제어 챔버에서 모기를 놓습니다. </li></ol> 참고 : 유전자 입을의 효율성이 높은 변수이며 세포와 기관에 의해 증명서 및 단백질 턴 오버 요금 및 dsRNA의 이해 효율을 포함한 여러 가지 요인에 따라 달라집니다. 우리는 입을는 1 일 게시 주입처럼 일찍 시작 것을 발견 가지고 있고, 최대 6 일 게시 주입에 마지막으로하실 수 있습니다. 입을를 확인하기 위해 모래 바닥 같은 양적 PCR과 서양 등 증명서와 단백질 검출 방법을 사용합니다. 참고 : 모기 주사와 RNAi - 중재 유전자 입을 기술의 여러 단계에 대한 몇 가지 변형이 있습니다. 일부 변형에 의해 설명되어 있습니다 : Boisson, 외 (2006)와 블랜딘, 외 (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>

유전자 입을의 효율성은 매우 다양하고 세포와 기관에 의해 증명서 및 단백질 턴 오버 요금 및 dsRNA의 이해 효율을 포함한 여러 가지 요인에 따라 달라집니다. 우리는 입을는 이르면 1로 일 게시 주사를 시작하고 최대 6 일 게시 주입 지난 수있는 것으로 나타났습니다. 입을를 확인하기 위해 모래 바닥 같은 양적 PCR과 서양 등 증명서와 단백질 검출 방법을 사용합니다.

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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>
</tr>		</tbody>
		</table>
		</div>

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

역방향 유전자 접근 방법은 모기에 벡터 병원균하는 유전자 underly 저항을 결정하는 매우 유용하게 입증했습니다. 이 비디오 프로토콜은 말라리아 기생충을 항구 아노 펠 레스 모기 gambiae로 dsRNA를 주입하기 위해 Dimo​​poulos 연구실에서 사용하는 방법을 보여줍니다. 사출 설정을 조작하고 흉부에 dsRNA를 주입 기술은 그림이다.

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

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 ...

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

Research

JoVE Journal

Biology

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

12.6K 조회수.  Johns Hopkins University. 역방향 유전자 접근 방법은 모기에 벡터 병원균하는 유전자 underly 저항을 결정하는 매우 유용하게 입증했습니다. 이 비디오 프로토콜은 말라리아 기생충을 항구 아노 펠 레스 모기 gambiae로 dsRNA를 주입하기 위해 Dimo​​poulos 연구실에서 사용하는 방법을 보여줍니다. 사출 설정을 조작하고 흉부에 dsRNA를 주입 기술은 그림이다.

비디오: 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.

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 ...

연구

생물학

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.

모스키토 리어링 (A. gambiae)에 대한 프로토콜

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.

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 ...

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 ...

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.

포유 동물 세포에서 RNAi 심사를위한 선교 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 Effectors의 세균성 배송 : Transkingdom RNAi

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 - 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 ...

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.

Drosophila melanogaster를위한 지구력 훈련 프로토콜과 세로 성능 Assays

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.

말라리아 모기 전송하기위한 다중 탐지 분석

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.

에 RNAi의 트리거 배달<em&gt; 아노 펠 레스 gambiae</em&gt; 번데기

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