모기 음식 : <ol><li> 음식 : 접지된 물고기 음식 (Aquaricare). 작은 핀치가 추가되어야합니다. </li><li> 식품 B : CAT 식품 (Purina)을 접지. 작은 핀치가 추가되어야합니다. </li><li> 식품 C : 고양이 음식 (Purina). 두 정제가 추가되어야합니다. </li></ol> 블러드 : 동물이나 인간의 혈액은 뒷면 모기하는 데 사용할 수 있습니다. 신선한 피가 주사기로 수집하여 혈액의 각 10 ML에 대한 CPD (항응고제)의 1ml를 포함하는 멸균 15 ML 팔콘 튜브에 넣어 부드럽게 섞어 5 분 2000 rpm으로 실온에서 centrifuged입니다. 뜨는 다른 혈액 세포 (예 : WBC)을 구성되어 버피 코트를 제거를 돌보는 삭제됩니다. RBC 펠렛은 다음 pipetting하여 RPMI 매체의 동일한 볼륨에 정지하고, 추가로이 중간에 세 번 씻어입니다. 최종 세척 후 pelleted RBCs가 RPMI 매체의 동일한 볼륨에 resuspended 아르 4에 저장된 ° C (80~10일 보존 수 있습니다.) 먹이를하기 전에, RBC는 (RPMI)를 셀 아래로 펠렛을 (5 분 2000 RPM) centrifuged이며, 포장 RBC는 40 % 혈소판을 얻기 위해 혈청 (O + 인간 혈청)에 resuspended 있습니다. 피를 ° C 항상 먹이를 위해 37 보관해야합니다. DAY 1 : <ol><li> 3-5일 오래된 성인 여성 모기는 알을 낳기 위해 혈액을 공급하고 있습니다. </li><li> 혈액 먹이를 들어, 인공 막 (parafilm) 수유 방법은 다음과 같이 사용됩니다 : <ol><li> 적혈구는 (혈액 준비 위 참조) ~ 40 %의 혈소판을 (RBC 40 % 혈청 60 %를 포장)을 구하는 열 inactivated 혈청과 혼합됩니다. 이것은 유리 피더에 추가됩니다. </li><li> 피더는 (37 ° C) 물 재킷 따뜻한 연결하고, 멤브레인 표면에 모기의 액세스를 허용하기 위해 새장에 배치됩니다. </li><li> 모기는 ~ 30 분 동안 피드를 사용할 수 있습니다. </li></ol></li></ol> 3 일 : <ol><li> 그들은 피를 먹이 후 암컷 이틀 알을 낳습니다. 원뿔 모양에 싼 작은 종이 필터는 필터 종이 촉촉한도록하고, 증류수가 들어있는 작은 비커에 들어가게된다. 비커는 알을 낳기 위해 모기에 대한 밤새 케이지 내부에 보관됩니다. </li></ol> DAY 4 : <ol><li> 모기 계란을 포함 여과지는 ~ 300 ML 증류수와 플라스틱 트레이에 배치됩니다. 음식의 핀치는 트레이에 추가하고 달걀은 다음 일 동안 애벌레로 부화 수 있습니다. </li></ol> 일 5-8 : <ol><li> 성장하는 애벌레가 음식을 C의 두 돌판과 함께 매일 공급하고, 밀도와 인구에 대한 모니터링입니다. 여덟째 날 (오일 된 애벌레)에서 애벌레 인구는 각 트레이에 식품 식품 C의 두 정제를 한 스푼과 함께 1 트레이에서 ~ 10 트레이로 희석합니다. (~ 30 분) </li></ol> 일 9-12 : <ol><li> 애벌레는 12 일 (구일 된 애벌레)에서 식품 C. 매일 같이 먹이 있으며, 물을 신선한 물로 변화이며, 음식 (식품 및 식품 B C의 두 정제의 핀치) 추가됩니다. pupae이 단계에서 개발 시작합니다. 쟁반은 성인의 탈출을 방지하기 위해 그물로 덮여있다. (~ 80-10 분) </li></ol> DAY 13-15 : 삼일 - pupae는 다음 2 성인에 등장할 수 있습니다. 식품은 신중하게 성인의 탈출 방지하기 위해 그물을 제거하여 애벌레 / pupae 매일 주어집니다. DAY 16 : <ol><li> 성인은 진공에 연결된 흡인기를 통해 케이지로 수집하고 있습니다. 케이지는 10 %의 자당 (autoclaved) 및 케이지 처리하는 동안 발생할 수있는 잠재적인 설탕 유출 소크하기 위해 바닥에 종이 타월 라이닝과 함께 배어있는 목화 심지와 작은 100 ML 병으로 구성되어 있습니다. (~ 30-40 분) </li></ol> DAY 17-21 : <ol><li> 성인 (남성과 여성 모두) 다음 4~5일에 대한 insectary 방에 보관들은 다음주기를 시작하기 위해 다시 혈액 공급되기 전에 10 %의 자당에 공급하고 있습니다. 동일한 모기는 한 번 이상 알을 낳기 위해 사용할 수 있습니다. </li><li> 실험이나 양육을 위해 필요하지 않은 모기는 냉동실에 새장을 배치에 의해 사망​​ 수 있습니다. 사용 트레이와 케이지 그들이 다시 사용하기 전에 청소하고 건조해야합니다. </li></ol> 참고 :이 모기 양육 방식에서 일부 차이를 보일 수 있으며, 다른 연구소 서로 다른 기술을 가지고 있습니다.

<ol>
	<li>Bangs, M. J., Soelarto, T., Barodji, B. P., Wicaksana, D. T., Boewono, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonization+of+Anopheles+maculatus+from+Central+Java,+Indonesia.">Colonization of Anopheles maculatus from Central Java, Indonesia.</a> J Am Mosq Control Assoc. 18, 359-363 (2002).</li><li>Beier, M. S., Beier, J. C., Merdan, A. A., el Sawaf, B. M., Kadder, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+rearing+techniques+and+adult+life+table+parameters+for+Anopheles+sergentii+from+Egypt.">Laboratory rearing techniques and adult life table parameters for Anopheles sergentii from Egypt.</a> J Am Mosq Control Assoc. 266, 266-270 (1987).</li><li>Benedict, M. Q., Crampton, J. M., Beard, C. B., Louis, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Care and maintenance of anopheline mosquitoes, The molecular biology of disease vectors: A methods manual. , 3-12 (1997).</li><li>Casanges, A. H., Mcgovran, E. R., Chiles, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rearing+of+Anopheles+quadrimaculatus+Say+and+Aedes+aegypti+(L.)+in+the+laboratory.">Rearing of Anopheles quadrimaculatus Say and Aedes aegypti (L.) in the laboratory.</a> Mosq News. 9, 112-117 (1949).</li><li>Merritt, R. W., Dadd, R. H., Walker, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeding+behavior,+natural+food+and+nutritional+relation+ships+of+larval+mosquitoes.">Feeding behavior, natural food and nutritional relation ships of larval mosquitoes.</a> Annual Rev. Entomol. 37, 349-376 (1992).</li><li>Nasirian, H., Ladonni, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+bloodfeeding+of+Anopheles+stephensi+on+a+membrane+apparatus+with+human+whole+blood.">Artificial bloodfeeding of Anopheles stephensi on a membrane apparatus with human whole blood.</a> J Am Mosq Control Assoc. 22, 54-56 (2006).</li><li>Ng'habi, K. R., John, B., Nkwengulila, G., Knols, B. G., Killeen, G. F., Ferguson, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+larval+crowding+on+mating+competitiveness+of+Anopheles+gambiae+mosquitoes.">Effect of larval crowding on mating competitiveness of Anopheles gambiae mosquitoes.</a> Malar J. 30 (4), 49 (2005).</li><li>Stobart, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+the+control+of+body+volume+in+the+larvae+of+the+mosquitoes+Aedes+aegypti+and+Aedes+detritus.">Factors affecting the control of body volume in the larvae of the mosquitoes Aedes aegypti and Aedes detritus.</a> Edw. J. Exp. Boil. 54, 67-82 (1971).</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>Food A</td>
<td>&nbsp;</td>
<td>Aquaricare</td>
<td>&nbsp;</td>
<td>Grounded fish food. A small pinch needs to be added.</td>
</tr>
<tr>
<td>Food B</td>
<td>&nbsp;</td>
<td>Purina</td>
<td>&nbsp;</td>
<td>Grounded cat food. A small pinch needs to be added.</td>
</tr>
<tr>
<td>Food C</td>
<td>&nbsp;</td>
<td>Purina</td>
<td>&nbsp;</td>
<td>Cat food. Two tablets needs to be added</td>
</tr>
<tr>
<td>RBC</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>prepared red blood cells: 40% hematocrit in human serum</td>
</tr>
<tr>
<td>RPMI</td>
<td>Medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CPD</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>anticoagulant</td>
</tr>
<tr>
<td>serum</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>O+ human serum</td>
</tr>
<tr>
<td>Mosquito feeder</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>Anopheles gambiae</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>mosquitos</td>
</tr>
<tr>
<td>10% sucrose</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>mosquito food</td>
</tr>
<tr>
<td>insectary</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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protocol for mosquito rearing (a. gambiae)

이 프로토콜은 insectary에 모기 양육에 대해 설명합니다. insectary 객실은 28 유지 ° C와 12 시간 함께 ~ 80 %의 습도. 주 / 야주기. 이 절차를 들어, 모기 연습장 10 % 살균 자당 솔루션, 종이 수건, 비커, 왓먼 필터 종이, 유리 모이통, 인간 혈액 및 혈청, 물 목욕, parafilm, 증류수, 깨끗한 플라스틱 쟁반, 모기 음식 (설명 필요 아래), 어른을 수집하기 위해 트레이, 진공 및 수집 챔버를 충당하기 위해 그물 모기.

Watch this Scientific Journal Video about Protocol for Mosquito Rearing A. gambiae at JoVE.com

Protocol for Mosquito Rearing A. gambiae

이 동영상은 실험실에서 뒤쪽 아노 펠 레스 gambiae하는 데 사용되는 일반적인 기법을 보여줍니다. 실험실 모기를 돌보는위한 방법은 혈액 먹이 어른 pupae에 애벌레에서 유기체의 생명주기의 모든 단계를 통해 증명하고 있습니다.

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 ...

Research

JoVE Journal

Biology

Protocol for Mosquito Rearing (A. gambiae)

23.1K 조회수.  Johns Hopkins University. 이 동영상은 실험실에서 뒤쪽 아노 펠 레스 gambiae하는 데 사용되는 일반적인 기법을 보여줍니다. 실험실 모기를 돌보는위한 방법은 혈액 먹이 어른 pupae에 애벌레에서 유기체의 생명주기의 모든 단계를 통해 증명하고 있습니다.

비디오: Protocol for Mosquito Rearing A. gambiae

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 ...

연구

생물학

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 ...

Neutrophil Isolation Protocol

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory immune response. Due to their key role in inflammation, neutrophil functions such as locomotion, cytokine production, phagocytosis, and tumor cell combat are extensively studied. To characterize the specific functions of neutrophils, a clean, fast, and reliable method of separating them from other blood cells is desirable for in vitro studies, especially since neutrophils are short-lived and should be used within 2-4 hours of collection. Here, we demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media that is a mixture of sodium metrizoate and Dextran 500. The procedure consists of layering whole blood over the density gradient medium, centrifugation, separation of neutrophil layer, and lysis of residual erythrocytes. Cells are then washed, counted, and resuspended in buffer to desired concentration. When performed correctly, this method has been shown to yield samples of >95% neutrophils with >95% viability.

Neutrophils are among the first cells to arrive on the site of inflammatory immune response, and their functions and mechanisms have been studied extensively in vitro. We demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media.

호중구 격리 프로토콜

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory ...

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia (SCG)

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide sympathetic innervations for the head and neck regions and they constitute a well-characterized and relatively homogeneous population (4).  Sympathetic neurons are dependent on nerve growth factor (NGF) for survival, differentiation and axonal growth and the wide-spread availability of NGF facilitates their culture and experimental manipulation (2, 3, 6).  For these reasons, cultured sympathetic neurons have been used in a wide variety of studies including neuronal development and differentiation, mechanisms of programmed and pathological cell death, and signal transduction (1, 2, 5, and 6).  Dissecting out the SCG from newborn rats and culturing sympathetic neurons is not very complicated and can be mastered fairly quickly.  In this article, we will describe in detail how to dissect out the SCG from newborn rat pups and to use them to establish cultures of sympathetic neurons.  The article will also describe the preparatory steps and the various reagents and equipment that are needed to achieve this.

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

This is a protocol describing how to isolate and culture primary sympathetic neurons from superior cervical ganglia (SCG) of newborn rat pups.

쥐 우수한 자궁암 신경 (SCG)에서 Culturing 교감 신경에 대한 프로토콜

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide ...

A Protocol for the Production of KLRG1 Tetramer

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. KLRG1 exists both as a monomer and as a disulfide-linked homodimer. This well-conserved receptor is found on the most mature and recently activated NK cells as well as on a subset of effector/memory T cells.
Using KLRG1 tetramer as well as other methods, E-, N-, and R-cadherins were identified as KLRG1 ligands. These Ca2+-dependent cell-cell adhesion molecules comprises of an extracellular domain containing five cadherin repeats responsible for cell-cell interactions, a transmembrane domain and a cytoplasmic domain that is linked to the actin cytoskeleton. 
Generation of the KLRG1 tetramer was essential to the identification of the KLRG1 ligands. KLRG1 tetramer is also a unique tool to elucidate the roles cadherin and KLRG1 play in regulating the immune response and tissue integrity.

This protocol describes the production of KLRG1 tetramer, which is a powerful tool for the analysis of KLRG1 ligands.

KLRG1 Tetramer의 생산을위한 프로토콜

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. ...

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine parasitoids known to infect the host during the egg stage1. Second, it has a wide range of potential fruit fly hosts. Perhaps due to its life history, F. arisanus has been a successfully used for biological control of fruit flies in multiple tropical regions2-4. One impediment to the wide use of F. arisanus for fruit fly control is that it is difficult to establish a stable laboratory colony5-9. Despite this difficulty, in the 1990s USDA researchers developed a reliable method to maintain laboratory populations of F. arisanus10-12. There is significant interest in F. arisanus biology13,14, especially regarding its ability to colonize a wide variety of Tephritid hosts14-17; interest is especially driven by the alarming spread of Bactrocera fruit fly pests to new continents in the last decade18. Further research on F. arisanus and additional deployments of this species as a biological control agent will benefit from optimizations and improvements of rearing methods. In this protocol and associated video article we describe an optimized method for rearing F. arisanus based on a previously described approach12. The method we describe here allows rearing of F. arisanus in a small scale without the use of fruit, using materials available in tropical regions around the world and with relatively low manual labor requirements.

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus is an egg-larval parasitoid of Tephritid fruit flies that is successfully used in biological control of these important tropical pests. We describe here an optimized protocol for rearing F. arisanus on a small scale using readily available materials.

리어링을위한 최적화된 프로토콜 Fopius arisanus Tephritid 과일 Parasitoid 난다

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

재조합 단백질의 미끼를 사용 파지 디스플레이와 궁합 선택을위한 프로토콜

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

SIVQ-LCM Protocol for the ArcturusXT Instrument

SIVQ-LCM is a new methodology that automates and streamlines the more traditional, user-dependent laser dissection process.&#160;It aims to create an advanced, rapidly customizable laser dissection platform technology. In this report, we describe the integration of the image analysis software Spatially Invariant Vector Quantization (SIVQ) onto the ArcturusXT instrument. The ArcturusXT system contains both an infrared (IR) and ultraviolet (UV) laser, allowing for specific cell or large area dissections. The principal goal is to improve the speed, accuracy, and reproducibility of the laser dissection to increase sample throughput. This novel approach facilitates microdissection of both animal and human tissues in research and clinical workflows.

SIVQ-LCM is an innovative approach that harnesses a computer algorithm, Spatially Invariant Vector Quantization (SIVQ), to drive the laser capture microdissection (LCM) process. The SIVQ-LCM workflow greatly improves the speed and accuracy of microdissection, with applications in both the research and clinical settings.

ArcturusXT 악기 SIVQ-LCM 프로토콜

SIVQ-LCM is a new methodology that automates and streamlines the more traditional, user-dependent laser dissection process.&#160;It aims to create an ...

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, most notably developmental arrest and reduced metabolism. Global gene expression profiling via RNA-Seq can provide important insights into the transcriptional mechanisms of photoperiodic diapause. The Asian tiger mosquito, Aedes albopictus, is an outstanding organism for studying the transcriptional bases of diapause due to its ease of rearing, easily induced diapause, and the genomic resources available. This manuscript presents a general experimental workflow for identifying diapause-induced transcriptional differences in A. albopictus. Rearing techniques, conditions necessary to induce diapause and non-diapause development, methods to estimate percent diapause in a population, and RNA extraction and integrity assessment for mosquitoes are documented. A workflow to process RNA-Seq data from Illumina sequencers culminates in a list of differentially expressed genes. The representative results demonstrate that this protocol can be used to effectively identify genes differentially regulated at the transcriptional level in A. albopictus due to photoperiodic differences. With modest adjustments, this workflow can be readily adapted to study the transcriptional bases of diapause or other important life history traits in other mosquitoes.

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data.

아시아 타이거 모기에 Photoperiodic 휴면의 RNA-서열 분석을위한 실험 및 생물 정보학 프로토콜,<em&gt; Aedes albopictus</em

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, ...

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 ...