作者感谢她的这个协议的批评维多利亚木匠。

第一部分- RNAi介导的基因敲除成年埃及伊蚊蚊。实验概述， 见图1。 1。双链RNA的合成<ol><li>对利益和控制的dsRNA基因合成的双链RNA。注：我们建议发展中国家引物的PCR片段范围在300至500个碱基对位于特定基因2月底3&#39;和T7引物序列在5&#39;端（5&#39;-TAA TAC GAC中TCA的CTA重视。豪GG-3&#39;）。片段的唯一性，应确认BLASTN程序分析3。 </li><li>使用的Ambion公司的T7 Megascript高产转录试剂盒（Ambion公司，试剂表），利用T7 RNA聚合酶转录反应合成双链RNA。注：类似的试剂和试剂盒可在别处。 </li><li>净化双链RNA，沉淀氯化锂遵循的Megascript套件指示。 </li><li>后purificat离子，在无菌水溶解的双链RNA沉淀。用于显微注射，以确保足够的粘度，dsRNA的浓度不得超过2微克/微升。 </li></ol> 2。注射液的制备<ol><li>可以构造一个简单的微注射器用管，剪刀，金属针，1 ml注射器，1毫升的塑料枪头（ 见图2）。应切断油管〜40厘米长。另外，自动化微注射器可用于如德拉蒙德Nanojet二4。 <ol><li> 2毫升的刻度切尖的注射器（针轮毂），并从橡胶柱塞头柱塞。 </li><li>打孔使用金属针在橡胶柱塞头和橡胶柱塞的后脑勺针枢纽。 </li><li>油管的一端放置在针枢纽，并将其放置在另一端1毫升的塑料枪头，将口片（另外，10毫升SYringe可用于生产注射所需的空气压力）。 </li></ol></li><li>孔中的橡胶柱塞头放置在一个玻璃毛细管针，并打破针尖所以宽度是足够大的液体流经。注：针尖的最优规模待定经验 - 如果针的宽度太大，这将导致创伤和高蚊子死亡率，如果针的宽度太小，这将是不可能的渗透蚊子的外骨骼。 </li><li>淹没在准备双链RNA样品注射针，并绘制成的注射针吸口片（或注射器）的液体，液体样品。注意：这一步是相同的所有液体试剂注入蚊子，包括PBS缓冲液，这是用来在体内利尿检测协议（见下文）。 </li></ol> 3。收集和麻醉蚊子<ol><li>收集用电池供电到一个收集瓶吸气ţ蚊子。放置在收集瓶的瓶盖和瓶放在一个干净的CO 2垫麻醉蚊子。注：另外，蚊子可以在冰麻醉。 </li></ol> 4。蚊子注射液<ol><li>打开收集瓶的CO 2垫直接放在蚊子，等到蚊子是麻醉。 </li><li>丢弃所有男性。 </li><li>排队上侧的蚊子，以便更容易地访问注射。 </li><li>抢蚊子的腿或翅膀，以避免受伤。你也可以用细的画笔或羽毛来操纵蚊子。 </li><li>当准备注入第一的蚊 ​​子，轻轻的支持与钳胸部一侧针尖插入对方胸部（ 图2）。这是更好地注入的角质层薄的部分，避免推针O连接到胸部深。 </li><li>一旦针到位，吹入蚊子的液体。所需的金额可以由监察针液体半月板。必要毫米为一个特定的卷的数目可以由玻璃毛细管针（πr2小时 ）的汽缸容积计算。通常用于注射用双链RNA的有效金额为1微克。 </li><li>一旦液体被注入到蚊子，仔细地缩回针头。如果胸部外大型液体液滴形式，蚊子应该被丢弃。然后，重复这个过程中，未来的蚊子。 </li></ol> 5。蚊子的恢复和存储<ol><li>注射后，放置在储存容器中的蚊子。例如，一品脱蜡内衬纸板杯（汤杯）网覆盖纸板盖保护。盖有网的覆盖暴露了削减部分。一旦所有的钼squitoes放置在容器中，容器放置在孵化室环境控制和提供食物来源，如20％蔗糖浸泡的棉花球放在网覆盖顶部的蚊子。 在体内利尿检测的，然后再执行，剥夺水源的双链RNA注射蚊子为12小时，以规范每个蚊子的水化状态。 </li><li>双链RNA介导的基因敲除效率可以是可变的。基因沉默可以开始注射后1天，它可能会持续注射后4至6天。确定的最佳时机，以实现最大的基因敲除每一个基因的经验。一般来说，我们等待双链RNA注射后的前3天进行。 </li></ol>第二部分- 在成人埃及伊蚊的蚊子体内利尿法 注：此协议已被发达国家和作者用于黄热病的蚊子的水通道蛋白介导的RNA干扰击倒的埃及伊蚊 。为了避免个别蚊子之间的变异，蚊子应分析组。由于技术上的原因，我们建议5％治疗蚊虫群体 - 有一个有限的时间内进行第一次测量体重，蚊子开始前，注射后尿排泄。 6。收集和麻醉蚊子<ol><li>之前收集的蚊子，记录与使用分析精密天平盖一个空的收集瓶的重量。这瓶将被用于所有后续测量。 </li><li>权衡收集瓶与抽气收集到5个雌性蚊子。将在收集瓶帽，让它坐几秒钟的CO 2垫麻醉蚊子。 </li></ol> 7。初始重量测量<ol><li>采取ţ他的5个蚊子初始体重测量，将收集瓶含有精密天平上的帽蚊子。 </li><li>计算组5蚊子的重量，通过蚊子的重量和收集瓶与盖和帽空收集瓶的重量减去。 </li><li>打开收集瓶和录制的蚊子的重量后，直接放置在CO 2垫蚊子。如果蚊子都开始醒来时体重测量，为几秒钟，然后打开它，并放在垫蚊子小瓶上的CO 2垫。 </li></ol> 8。注射液的制备<ol><li>设立微注射器RNAi介导的基因敲除协议中的指示。 </li><li>放置一个玻璃毛细管在微注射器针头和打破针尖宽度足够大的液体uid来流经。 </li><li>淹没的针在PBS缓冲液和注射针吸引到缓冲区，使用此协议所需的金额是1.25每个蚊子的PBS液。注：此金额模仿，采取雌蚊5血浆的平均金额。 </li></ol> 9。蚊子注射液<ol><li>排队的蚊子，以便更容易与微注射器的访问。 </li><li>一旦针到位，吹PBS缓冲液，把蚊子。 </li><li>一旦液体被注入到蚊子，可能形成液滴在胸部外侧。这液滴下一步前要认真清除。 </li><li>重复注射过程中，与未来的蚊子。随着经验的积累，蚊子的存活率会是注射后几乎100％。 </li></ol> 10。称重蚊子<ol><li>注射后，轻轻将蚊子在收集瓶和帽子。 5蚊子将收集瓶含有精密天平上的帽蚊子，以第一次测量体重。 </li><li>蚊子和收集瓶与盖的重量计算组5蚊子的重量减去空收集瓶与盖的重量。注：蚊子会开始分泌尿液中的CO 2麻醉垫取出后2分钟内，所以重要的是要测量体重才开始排泄。 </li><li>放置在一个小容器中的蚊子，他们将在那里开始排泄尿液。 </li></ol> 11。第二个和随后的体重测量 注：在30分钟的时间间隔应采取的蚊子的重量测量，但可以调整或长或短的时间间隔取决于排泄率。 <ol><li> 30分钟后，收集石斑鱼页5抽吸到相同的收集瓶与盖蚊子。采取将收集瓶含有精密天平上的帽蚊子蚊子的下一个体重测量。 </li><li>测量后，将在接下来的30分钟在同一控股容器的蚊子。 </li><li>重复这个过程所需的时间量。 </li></ol> 12。确定蚊子排泄率<ol><li>可以计算出被注入到5蚊子的液体总量减去初始体重的重量后，立即注射蚊子。 </li><li>可以计算出在一个特定的时间点是由蚊子组排出的尿液量减去初始体重从重量在特定时间点的蚊子蚊子。 </li><li>在一个特定时间点的排泄率，可以计算出航空维修丁在这个时间点尿排出的液体注入（ 见表1）的总金额的数额。 </li></ol> 13。代表结果 RNAi介导的基因敲除和体内利尿法已用于汉森实验室研究水通道蛋白在埃及伊蚊利尿的效果。三是水通道蛋白表达在埃及伊蚊马氏管被撞倒图4显示了代表利尿法已被用来排泄率比较埃及伊蚊和致倦库蚊实验结果相比，控制蚊子1排泄率显着效果。在不同的温度。 <img src="/files/ftp_upload/3479/3479fig1.jpg" alt="图1" /> 图1。 RNA干扰/利尿法流程图 5组，每组10蚊子EA。CH注射dsRNA的一个特定的基因，十蚊五组与对照双链RNA注入。另一组200μM2 HgCl在PBS注射蚊子被用来作为阳性对照。这些蚊子注射前和注射3小时30分钟的时间间隔后称重。 <img src="/files/ftp_upload/3479/3479fig2.jpg" alt="图2" /> 图2。 RNAi介导的基因敲除和在体内利尿法的一个简单的微注射装置。A。用于注射的玻璃毛细管针。灰色的三角形代表毫米针提请表明液体注射到蚊子的数量递增。乙1毫升注射器用于兴建微注射器。白色三角形代表针枢纽和黑色三角形代表的橡胶柱塞头连接在注射器的柱塞。 C.油管用来连接的喉舌喷油器。 D. 1毫升一次性吸管头（蓝色提示）的显微注射设备的喉舌。 E.显微注射设备，采用公元部分。 <a href="http://www.jove.com/files/ftp_upload/3479/3479fig2large.jpg" target="_blank">点击这里查看大图</a> 。 <img src="/files/ftp_upload/3479/3479fig3.jpg" alt="图3" /> 图3。最佳蚊子注射部位。答与胸部之间的大尺度的玻璃毛细管针注射雌性埃及伊蚊 。黑条表示1毫米大小的比较。 B.绘图的雌蚊胸部和白色斑点代表在蚊子外骨骼的白色鳞屑。注射针应刺破景点之间的蚊子，以尽量减少注射时的死亡率。 <img src="/files/ftp_upload/3479/3479fig4.jpg" alt="图4" /> 图4。对温度的影响致倦库蚊和埃及伊蚊利尿TURE。利尿法两蚊， 埃及伊蚊和致倦库蚊种，在不同温度下进行。在注射后的第一个小时的排泄率（％）。 <table border="1"><tbody><tr><td> 组 </td><td> 塔拉 （七） </td><td> 不注射 （七） </td><td> 注射后 （七） </td><td> 后1h进样 （七） </td><td> 平均体重（毫克） </td><td> 量注射 （μL） </td><td> 金额排出 （μL） </td><td> ％排泄 </td></tr><tr><td> 1 </td><td> 7.5938 </td><td> 7.6057 </td><td> 7.6104 </td><td> 7.6096 </td><td> 2.38 </td><td> 0.94 </td><td> 0.16 </td><td> 17.0 </td></tr><tr><td> 2 </td><td> 7.8252 </td><td> 7.8349 </td><td> 7.8415 </td><td> 7.8403 </td><td> 1.94 </td><td> 1.32 </td><td> 0.24 </td><td> 18.2 </td></tr><tr><td> 3 </td><td> 7.8896 </td><td> 7.9026 </td><td> 7.9077 </td><td> 7.906 </td><td> 2.6 </td><td> 1.02 </td><td> 0.34 </td><td> 33.3 </td></tr></tbody></table> 表1。 埃及伊蚊在执行与埃及伊蚊雌蚊在体内利尿法在4°C的原始数据体内利尿检测结果。

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我们什么都没有透露。

RNAi的协议，用于已开发在Raikhel亚历山大实验室在加州河滨大学6,7和类似由加弗和Dimopoulos 4发表了一份协议。这个视频协议中的实验方法可以用来研究有关的基因在昆虫体内设置利尿。作为一个简单的“利尿模型系统，吸引昆虫，马氏管，排泄器官几代科研人员的利益。这个器官是涉及通关8和果蝇的工作已确定在马氏管生理9,10，现在可以在其他昆虫...

<table border="1"><tbody><tr><td> 试剂或设备名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> MEGAscript的T7高产套件</td><td> Ambion公司，公司</td><td> AM1334 </td><td></td></tr><tr><td> PBS缓冲液</td><td> Sigma-Aldrich公司</td><td> P4417 </td><td></td></tr><tr><td>塑料管材</td><td>本地供应商</td><td></td><td>聚氯乙烯</td></tr><tr><td> 1毫升胶枪头</td><td>厂商VWR </td><td> 83007-376 </td><td>蓝尖</td></tr><tr><td> 1毫升注射器</td><td>流式细胞，Dickinson公司</td><td> 309602 </td><td></td></tr><tr><td>剪刀</td><td>本地供应商</td><td></td><td></td></tr><tr><td>金属针</td><td>卡罗来纳州生物</td><td> 654307 </TD&gt; <td>大小5 </td></tr><tr><td>飞垫</td><td>杰纳西科学</td><td> 789060 </td><td></td></tr><tr><td>电池供电的吸气/收集瓶</td><td> UPMA实验室</td><td> IPMM 2000 </td><td></td></tr><tr><td>细尖镊子</td><td>世界精密仪器</td><td> 14095 </td><td></td></tr><tr><td>玻璃毛细管针</td><td>世界精密仪器</td><td> 1B200-6 </td><td></td></tr><tr><td>立体解剖显微镜</td><td>徕卡公司</td><td> S6D </td><td></td></tr><tr><td>分析精密天平</td><td>梅特勒 - 托利多</td><td> AB54S </td><td></td></tr><tr><td>蔗糖</td><td> Sigma-Aldrich公司</td><td> 84097 </td><td></td></tr><tr><td>一品脱打蜡内衬纸板杯</td><td>本地供应商</td><td></td><td>制造汤杯</td></tr><tr><td>网状网</td><td>本地供应商</td><td></td><td>塑料飞纱布的</td></tr></tbody></table>

rnai-mediated gene knockdown and in vivo diuresis assay in adult female aedes aegypti mosquitoes

这个视频协议演示了一个有效的技术击倒在昆虫特定基因，并进行一种新的生物活性来衡量排泄率。这种方法可以用于获取更好地了解昆虫的利尿过程，是在利尿吸血节肢动物，能够占用大量的液体，在一个单一的血粉的研究，特别是有用的。 这RNAi介导的基因敲除结合在体内利尿法是由汉森实验室研究RNAi介导水通道蛋白基因埃及伊蚊利尿1击倒的影响。 该协议是在设置两部分：第一个示范说明如何构建一个简单的蚊子喷射装置及如何准备和注入的蚊子胸部RNAi介导的基因敲除的dsRNA。第二个示范说明如何确定在使用延命蚊子的排泄率。

Watch this Scientific Journal Video about RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes at JoVE.com

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

在这个协议中，我们结合RNAi介导的基因沉默<em&gt;在体内</em&gt;利尿法研究感兴趣的基因的影响击倒蚊虫液的排泄。

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 ...

rnai-mediated-gene-knockdown-vivo-diuresis-assay-adult-female-aedes

Research

JoVE Journal

Biology

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

13.4K 次观看.  New Mexico State University. 在这个协议中，我们结合RNAi介导的基因沉默在体内利尿法研究感兴趣的基因的影响击倒蚊虫液的排泄。

视频: RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

Injection of dsRNA into Female A. aegypti Mosquitos

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 James lab to inject dsRNA into female A. aegypti mosquitoes, which harbor the dengue virus.  The technique for calibrating injection needles, manipulating the injection setup, and injecting dsRNA into the thorax is illustrated.

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 James lab to inject dsRNA into female A. aegypti mosquitoes, which harbor the dengue virus. The technique for calibrating injection needles, manipulating the injection setup, and injecting dsRNA into the thorax is illustrated.

女答：埃及伊蚊蚊的dsRNA注入

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video ...

科研

生物学

Microinjection of A. aegypti Embryos to Obtain Transgenic Mosquitoes

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever.  The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever. The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

埃及斑蚊胚胎显微注射获得转基因蚊子

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

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 ...

Dissection of Midgut and Salivary Glands from Ae. aegypti Mosquitoes

The mosquito midgut and salivary glands are key entry and exit points for pathogens such as Plasmodium parasites and Dengue viruses. This video protocol demonstrates dissection techniques for removal of the midgut and salivary glands from Aedes aegypti mosquitoes. 



The mosquito midgut and salivary glands are key entry and exit points for vector pathogens like Plasmodium falciparum and the dengue virus. This video demonstrates the dissection techniques for removing the midgut and salivary glands from Aedes aegypti mosquitoes.

从AE肠和唾液腺的解剖。埃及伊蚊蚊

The mosquito midgut and salivary glands are key entry and exit points for pathogens such as Plasmodium parasites and Dengue viruses. This video protocol ...

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

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite.  The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Protocol for RNAi Assays in Adult Mosquitoes A. gambiae

Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite. The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

成蚊（冈比亚按蚊的RNAi实验议定书）

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans3, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis4 and Pristionchus pacificus5. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior6-7, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers5. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis4 and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms8. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F1 progeny. Unlike C. elegans, but like most other nematodes, P. pacificus PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4 promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92) mutant in a visually informative protocol.

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

In model organisms, transgenesis can manipulate gene functions while RNAi can knockdown specific mRNA transcripts 1-2. This protocol aims to illustrate the techniques needed to introduce stably transmitted DNA and transient double stranded RNA into the necromenic nematode Pristionchus pacificus for studies in evolutionary, developmental, and behavioral biology.

通过显微注射介导的RNAi Necromenic线虫基因敲除和转基因 Pristionchus pacificus</em

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression ...

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter implantation. The technical limitation of this surgical technique lies in the finesse of the hands. The injection is rapid, especially for a trained experimenter, and since tissue disruption with this technique is minimal, repeated injections are possible; moreover immune reaction to foreign tools (e.g. catheter) does not occur, thereby giving a better and more specific read out of spinal cord modulation. Since the application of the substance is largely limited to the target region of the spinal cord, drugs do not need to be applied in large dosages, and more importantly unwanted effects on other tissue, as observed with a systemic delivery, could be circumvented1,2. Moreover, we combine this technique with in vivo transfection of nucleic acid with the help of polyethylenimine (PEI) reagent3, which provides tremendous versatility for studying spinal functions via delivery of pharmacological agents as well as gene, RNA, and protein modulators.

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a simple and rapid technique of intrathecal needle puncture for a localized transfection of siRNA in the lumbar spinal cord in mouse under short lasting light anesthesia.

体内转染和基因敲除在脊髓通过快速无创腰椎鞘内注射小鼠

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter ...

Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus

The insect fat body plays a central role in insect metabolism and nutrient storage, mirroring functions of the liver and fat tissue in vertebrates. Insect fat body tissue is usually distributed throughout the insect body. However, it is often concentrated in the abdomen and attached to the abdominal body wall.
The mosquito fat body is the sole source of yolk proteins, which are critical for egg production. Therefore, the in vitro culture of mosquito fat body tissues represents an important system for the study of mosquito physiology, metabolism, and, ultimately, egg production. The fat body culture process begins with the preparation of solutions and reagents, including amino acid stock solutions, Aedes physiological saline salt stock solution (APS), calcium stock solution, and fat body culture medium. The process continues with fat body dissection, followed by an experimental treatment. After treatment, a variety of different analyses can be performed, including RNA sequencing (RNA-Seq), qPCR, Western blots, proteomics, and metabolomics.
In our example experiment, we demonstrate the protocol through the excision and culture of fat bodies from the yellow fever mosquito, Aedes aegypti, a principal vector of arboviruses including dengue, chikungunya, and Zika. RNA from fat bodies cultured under a physiological condition known to upregulate yolk proteins versus the control were subject to RNA-Seq analysis to demonstrate the potential utility of this procedure for investigations of gene expression.

Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus

The fat body is the central metabolic organ in insects. We present a live organ culture system that enables the user to study the responses of isolated fat body tissue to various stimuli.

肥胖的身体器官培养体系中蚊，一个向量 Zika 病毒

The insect fat body plays a central role in insect metabolism and nutrient storage, mirroring functions of the liver and fat tissue in vertebrates. Insect ...

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, chikungunya and Zika. Wolbachia-infected mosquitoes can be released into the field to either reduce population sizes through incompatible matings or to transform populations with mosquitoes that are refractory to virus transmission. For these strategies to succeed, the mosquitoes released into the field from the laboratory must be competitive with native mosquitoes. However, maintaining mosquitoes in the laboratory can result in inbreeding, genetic drift and laboratory adaptation which can reduce their fitness in the field and may confound the results of experiments. To test the suitability of different Wolbachia infections for deployment in the field, it is necessary to maintain mosquitoes in a controlled laboratory environment across multiple generations. We describe a simple protocol for maintaining Ae. aegypti mosquitoes in the laboratory, which is suitable for both Wolbachia-infected and wild-type mosquitoes. The methods minimize laboratory adaptation and implement outcrossing to increase the relevance of experiments to field mosquitoes. Additionally, colonies are maintained under optimal conditions to maximize their fitness for open field releases.

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes infected with Wolbachia are being released into natural populations to suppress the transmission of arboviruses. We describe methods to rear Ae. aegypti with Wolbachia infections in the laboratory for experiments and field release, taking precautions to minimize laboratory adaptation and selection.

维护伊蚊蚊蚊子感染沃尔巴克

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, ...

Improved Fecundity and Fertility Assay for Aedes aegypti using 24 Well Tissue Culture Plates (EAgaL Plates)

Mosquitoes represent a significant public health problem as vectors of various pathogens. For those studies that require an assessment of mosquito fitness parameters, in particular egg production and hatch rates at the individual level, conventional methods have put a substantial burden on investigators due to high labor intensity and laboratory space requirements. Described is a simple method using 24 well tissue culture plate with agarose in each well and digital imaging of each well to determine egg numbers and hatch rates at an individual level with substantially reduced time and space requirements.

Improved Fecundity and Fertility Assay for Aedes aegypti using 24 Well Tissue Culture Plates EAgaL Plates

Described is a time and space-saving method to count eggs and determine hatch rates of individual mosquitoes using 24 well tissue culture plates, which can substantially increase the scale and speed of fecundity and fertility assays.

使用 24 个井组织培养板 （EAgaL 板） 改进 了伊蚊 的丰度和生育率测定

Mosquitoes represent a significant public health problem as vectors of various pathogens. For those studies that require an assessment of mosquito fitness ...