这项研究由美国国立卫生研究院授予 #SC1AI109055，支持 2014年的设计 HHMI 补助金 #52008103 和 NSF PGR 补助金 #1238731。我们感谢与会者设计春天 2015 BIOL302 分子方法类和地位巴蒂亚的脂肪体文化实验技术支持。

1.制备溶液和试剂 <ol><li>氨基酸原液 <ol><li>准备 4 X 氨基酸原液 23 ， 24 用称重和20 不同氨基酸添加浓度在 表 1 中给出的锥形瓶。 
		注: 在某些情况下，amino acid(s) 可以排除在解决，取而代之的是等摩尔量的甘露醇，以保持平等的渗透调节物质含量。</li>
		<li>添加适量的 ddH 2 O，生产所需的数量的解决方案。</li>
		<li>以确保，氨基酸有完全溶解，文火加热时不断搅拌，直到液体已完全清除; 它将作出轻微的黄色色调。 
		注意: 它可能需要降低 ph 值至 6 由加入盐酸，允许一些更加疏水性氨基酸，解散。</li>
		<li>分装解决方案和无菌过滤使用注射器过滤器与 0.2 µ m 孔径大小。</li>
		<li>存储在密封的容器，在-20 ° C 至 6 个月。</li>
	</ol></li> <li>AP 注: 见 第 25。
	<ol><li>20 X 盐原液，结合 表 2 中列出在 50 毫升的水中的盐。搅拌，直到完全溶解。 
		注意: 它可能需要稍热解决方案完全溶解盐。</li>
		<li>无菌过滤器针头式过滤器用 0.2 微米孔径大小和存储解决方案在 50 毫升筒在-20 ° C.</li>
	</ol></li> <li>钙股票解决方案 <ol><li>X 钙股票溶液 50，将 0.90 g 氯化钙的添加到 100 毫升的水 (表 3); 搅拌，直到完全溶解。</li>
		<li>无菌过滤器使用注射器带通滤波器的 0.2 微米孔隙大小，分装解决方案到 50 毫升管，并储存在-20 ° C.</li>
	</ol></li> <li>三缓冲区 (pH 7.4) <ol><li>为招聘缓冲，盐与 表 4 中列出的解决方案相结合，并添加 ddH 2 O 达 100 mL。</li>
		<li>无菌过滤器使用注射器过滤 0.2 微米孔隙大小与分装进 50 毫升管溶液; 储存在室温。</li>
	</ol></li> <li>脂肪体培养基 <ol><li>准备脂肪体培养基，准备上面列出的所有解决方案。</li>
		<li>结合他们根据 表 5，使 200 毫升的脂肪体培养基中卷。</li>
		<li>调节 pH 至 7.2 使用 NaOH 或 HCl。</li>
		<li>无菌过滤解决方案使用注射器过滤器与 0.2 微米孔隙大小。</li>
		<li>解决方案分为 15 毫升和储存在-20 ° C，6 个月。</li>
	</ol></li></ol> 2。准备清扫 <ol><li>准备体视显微镜 （10-20 倍放大） 的照明设备和布置两个超精细镊子和一把剪刀微解剖。</li>
	<li>准备所有解决方案所需的实验。解冻至室温的脂肪体培养基。 
	注意: 沉淀能溶解的轻轻该溶液加热到不高于 30 ° C.</li>
	<li>与吸气，收集成年雌性蚊子 （3-7 天后出现） 和麻醉他们使用二氧化碳或冰 
	注意: 一旦麻醉，蚊子应该根据 CO 2 最短的时间可能不超过 20 分钟，或者，蚊子可以在冰上麻醉和保持大约 30 分钟</li>
	<li>准备与 AP 每口井的 3 毫升 6 孔板。</li>
</ol> 3。脂肪体夹层 <ol><li>集中夹层体视显微镜和调节到舒适的高度椅子。</li>
	<li>凹玻片置于显微镜表面和向中心添加两滴 APS。</li>
	<li>的一条腿，用镊子拿起一只蚊子并将它转移到 APS 表面显微镜幻灯片上。</li>
	<li>仔细紧抓蚊子的腹部，左手，钳和旋转蚊子，使腹边向上。</li>
	<li>同时胸部保持身体稳定，用镊子在右手里，抓住最后两个腹段，并将轻轻拉。 
	注: 腹部的最后两段分开将腹部和附加的卵巢、 马氏管、 后肠和中肠;有时这种作物会滑出腹部。如果他们不被删除，提示关门的钳插入小孔创建和删除其余组织。</li>
	<li>设置右钳放在一边，拿起弹簧剪刀。一个刀片式服务器滑进洞腹部到胸部以下的部分。轻轻地、 迅速地腹部纵向切。 
	注意: 一旦取得了伤口，腹部应该开始扩大向外，虽然这可能不会立即发生。</li>
	<li>继续进行第二个切口，将横向切割低于胸部，略低于第一次切割结束的地方。 
	注: 腹部应该开放然后从胸部上的角质层与脂肪体侧沉浸在 APS 游离。这腹壁的身体是 体外 培养的脂肪体准备。</li>
	<li>电梯从 AP 解决方案使用的镊子尖端脂肪体和将它们从溶液转移到含 AP 在室温的 6 孔板。允许组织推进到脂肪体文化之前休息大约 0.5 h。</li>
</ol> 4。肥胖的身体文化 <ol><li>孵育 AP 上的脂肪体，在室温下至少 0.5 h 来平衡他们。</li>
	<li>传输使用的钳尖端的脂肪体和轻轻地将它们从 AP 溶液。降低提示脂肪体培养液中。 
	注: 脂肪体应开放介质的表面上和自由浮动。肥胖的身体文化，通常会在 96 孔板，与介质在每口井的 150-200 微升。一个可以适合达三个人脂肪体，和他们都超过 12 小时 （未发表个人结果） 并不可行。</li>
	<li>后潜伏期，转化为脂肪体从介质中包含适当的试剂为下游加工的 1.5 mL 离心管。 
	注: 典型的分析包括 qPCR 16 ， ， 17 18 ， 19，印迹 18 ， 20、 26 转录组学、 蛋白质组学 21 或代谢组学 22。</li>
</ol>

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+to+determine+cell+viability+by+simultaneous+staining+with+fluorescein+diacetate-propidium+iodide.">An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide.</a> J Histochem Cytochem. 33 (1), 77-79 (1985).</li><li>Strober, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypan+blue+exclusion+test+of+cell+viability.">Trypan blue exclusion test of cell viability.</a> Curr Protoc Immunol. A3B, (2001).</li></ol>

我们没有什么可披露。

广泛用于昆虫器官培养研究昆虫内分泌学、 发展和代谢功能，探讨特定的器官与共生细菌29，，3031，之间的相互作用 32，，3334。体外脂肪体器官培养专门用于研究氨基酸运输及卵黄蛋白生产的蚊子和其他双翅目16，，1735规例 》,36.在卵黄发生的过程中，蚊子脂肪体使用数组的特异性高氨基酸转运载体从血淋巴，合成大量的卵黄蛋白12 导入血粉衍生的氨基酸19，，3536。肥胖的身体文化是有助于在这方面18的脂肪体营养要求划定。
起始物料，雌性蚊子，质量是这些实验成功的关键。在下拥挤的环境中成长和通常对高营养饲喂蚊子幼虫产生最好的结果。有一些重要的变量，建立蚊子脂肪体文化条件在实验室在实验设计时要考虑。我们在以往的研究脂肪体基因表达存在显著差异取决于个人的生活史和营养状况的蚊子11，22展示。蚊子文化条件应该是一致的以减少大小的变化程度和实验蚊子的营养储备。此外，应培训人员执行夹层动脉瘤，以确保快速、 准确，夹层动脉瘤的结果一致。可以使用不同染色方法37，38检查孤立脂肪体内细胞活力。
肥胖的身体文化实验的实验设计应考虑到夹层动脉瘤的数量可能在给定的时间段。需要大量的脂肪体时，可能需要多个夹层会话或多个解剖器。有是今后申请体外脂肪体文化在蚊子和其他昆虫的种类繁多。它将用于测试潜在的候选药物来控制昆虫尤其有用。转基因技术在昆虫来表达特定的记者蛋白质脂肪体滋养层细胞中的使用将开放发展强大生物鉴定为脂肪体生理研究的新方法。

蚊子是破坏性的人类疾病，包括疟疾、 登革热、 基孔肯亚热和 Zika1，，23向量。尽管激烈的国际努力来制止这些疾病的发生，控制传播疾病的蚊子，蚊子传染的疾病疫情是仍很常见，尤其是在发展中国家。有效的疫苗对抗许多这类疾病是不可用或效力有限的4，5。防止疫情的最有效途径是控制蚊子种群，主要使用杀虫剂处理。然而，杀虫剂的抗药性已在许多蚊子种群发展，成为一个共同的问题，围绕世界6，，78。蚊子生理学研究的新型工具和战略，以控制疾病的发展至关重要。
蚊子脂肪体贮藏营养、 代谢平衡、 复制和异生分解代谢9，10，，1112中央的作用。它是甘油三酯、 糖原和形式的贮藏蛋白质中氨基酸的主要存储机构。它也可以作为大多数血淋巴蛋白和代谢产物合成的位置。肥胖的身躯在蚊子之后他们采取血粉13，14, 发生在女性的卵黄蛋白生产的唯一来源。
肥胖的主要细胞类型是身体的大型、 多倍体的滋养细胞或脂肪细胞3，9，，1012。脂肪体组织组织成裂片或鞘和可以见于所有身体部位的蚊子，与最大的部分，位于的腹部的脂肪体大裂片相连的身体腹壁。
这里介绍的蚊子脂肪体文化系统开发了在 70 年代和仍然是研究肥胖的身体生理10，尤其是在当前分析技术结合的有力工具。这种技术的基础基于身体腹壁和关联的脂肪体组织的隔离。腹部的角质层的疏水性质使其漂浮在培养基中，与附加的裂片的沉浸的腹部脂肪体的表面。气门和 tracheolar 结构进行维护，确保培养组织氧合。今后，我们将这些准备工作称为"脂肪体"。孤立的脂肪体存活超过 12 小时时孵化在适当的媒介 （未发表的结果）。脂肪体文化是一个宝贵的工具，已解决各种问题关于脂肪体内分泌学和生理学9，，1012，15，16， 17。
体外培养的脂肪体可以遭受各种实验和控制的处理，可由调查员后决定的时间。在年底的潜伏期，脂肪体可以收集和处理下游的分析，包括 qPCR16，，1718，19，印迹18 20、 蛋白质组学21或代谢组学22。可以在不同的尺度，从群体可以共同培养的几百个人脂肪体上执行实验。
这里包括代表结果来自脂肪体培养氨基酸和类固醇激素 20-羟基蜕皮激素来模拟卵黄16，17，血粉激活， 2324。我们进行了分析和比较基因差异表达的不激活与活化脂肪体通过下一代测序分析。

<table border="0" cellpadding="0" cellspacing="0" width="508">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:64px;">Scissors&#160;</td>
			<td style="width:192px;">Fiskars</td>
			<td style="width:143px;">83872</td>
			<td style="width:111px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:64px;">Fly pad</td>
			<td style="width:192px;">Genesee Scientific</td>
			<td style="width:143px;">789060</td>
			<td></td>
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:64px;">Battery-powered aspirator w/ collection vial</td>
			<td style="width:192px;">Hausherrs Machine Works, Inc.</td>
			<td style="width:143px;">3740-01-210-2368</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:64px;">Fine tip forceps</td>
			<td style="width:192px;">World Precision Instruments, Inc.&#160;</td>
			<td style="width:143px;">500085</td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:64px;">Light microscope&#160;</td>
			<td style="width:192px;">Leica Microsystems</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:64px;">96 well plate&#160;</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:143px;">CL S3383</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:64px;">Sucrose</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:143px;">S9378</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Alanine</td>
			<td style="width:192px;">Sigma</td>
			<td>A7627</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Arginine</td>
			<td style="width:192px;">Sigma</td>
			<td>A5006</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Asparagine</td>
			<td style="width:192px;">Sigma</td>
			<td>A0884</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Aspartic Acid</td>
			<td style="width:192px;">Sigma</td>
			<td>A9256</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Cysteine</td>
			<td style="width:192px;">Sigma</td>
			<td>W326305</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Glutamic Acid</td>
			<td style="width:192px;">Sigma</td>
			<td>G1251</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Glutamine</td>
			<td style="width:192px;">Sigma</td>
			<td>G3126</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Glycine</td>
			<td style="width:192px;">Sigma</td>
			<td>G2879</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Histidine</td>
			<td style="width:192px;">Sigma</td>
			<td>H6034</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Isoleucine</td>
			<td style="width:192px;">Sigma</td>
			<td>I2752</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Lysine</td>
			<td style="width:192px;">Sigma</td>
			<td>L5501</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Leucine</td>
			<td style="width:192px;">Sigma</td>
			<td>L8000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Phenylalanine</td>
			<td style="width:192px;">Sigma</td>
			<td>P2126</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Proline</td>
			<td style="width:192px;">Sigma</td>
			<td>P0380</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Serine</td>
			<td style="width:192px;">Sigma</td>
			<td>S4500</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Threonine</td>
			<td style="width:192px;">Sigma</td>
			<td>T8625</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Tryptophan</td>
			<td style="width:192px;">Sigma</td>
			<td>T0254</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Tyrosine</td>
			<td style="width:192px;">Sigma</td>
			<td>T3754</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Valine</td>
			<td style="width:192px;">Sigma</td>
			<td>V0500</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Methionine</td>
			<td style="width:192px;">Sigma</td>
			<td>M9625</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:64px;">NaCl</td>
			<td style="width:192px;">Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:64px;">KCl</td>
			<td style="width:192px;">Sigma</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:64px;">MgCl2-6H2O</td>
			<td style="width:192px;">Sigma</td>
			<td>M2670</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:64px;">NaHCO3</td>
			<td style="width:192px;">Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:64px;">CaCl2-2H2O</td>
			<td style="width:192px;">Sigma</td>
			<td>C8106</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:64px;">Tris pH8.0&#160;</td>
			<td style="width:192px;">Sigma</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:64px;">EDTA</td>
			<td style="width:192px;">Sigma</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:64px;">ddH2O</td>
			<td style="width:192px;">Sigma</td>
			<td>W4502</td>
			<td></td>
		</tr>
	</tbody>
</table>

fat body organ culture system in aedes aegypti, a vector of zika virus

昆虫的脂肪体扮演主角在昆虫代谢和营养存储，镜像功能的肝脏和脂肪组织中的脊椎动物。昆虫的脂肪体组织通常分布在昆虫全身。然而，它往往是集中在腹部和附加到身体腹壁。
蚊子脂肪体是关键产蛋的蛋黄蛋白的唯一来源。因此，蚊子脂肪体组织的体外文化代表的蚊子生理学研究的重要制度代谢，并最终产蛋。肥胖的身体文化过程始于溶液和试剂，包括氨基酸股票解决方案、白纹伊蚊盐股票生理盐水 (APS)、 钙股票解决方案，以及脂肪体培养基的制备。脂肪体夹层，其次是实验性的治疗继续该过程。治疗后，可以执行各种不同的分析，包括 RNA 序列 (RNA Seq)、 qPCR、 西方墨水、 蛋白质组学、 代谢组学。
在我们的示例实验中，我们说明了通过切除和脂肪体文化协议从黄热病蚊子，蚊，虫媒病毒包括登革热、 基孔肯亚热和 Zika 主矢。从已知的上调蛋黄蛋白与控制生理条件下培养的脂肪体 RNA 受到 RNA 序列分析，表明本程序用于基因表达调查潜在的效用。

作为一个例子，我们进行了脂肪体文化实验和刺激孤立的脂肪体通过孵化它们对一个包含所有二十种天然氨基酸和昆虫类固醇激素 20-羟基蜕皮 (10 µ M) 为 6 h 的平衡的混合物的解决方案.作为对照，脂肪体孵育在 AP 上相当长的时间。
孵化后总 RNA 是孤立使用三试剂27按照制造商的说明。提取的 RNA 样品的质量进行评估使用分光光度法、 荧光定量和琼脂糖凝胶电泳。RNA 序列库使用 4 μ g 的总 RNA 生成和进行量化使用两个不同的技术。随后，图书馆被送往商业供应商配对末端测序。
这次实验的结果是表 6所示。基因显示对氨基酸和 20-羟基蜕皮酮的最强转录反应是主要卵黄蛋白基因，与以前的结果11。
图 1显示热地图指示的 1,256 基因的表达水平差异表达基因片段体外培养脂肪体后两种不同方法治疗。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55508/55508fig1.jpg"> 
图 1.热图，基因表达在脂肪体文化。基于特定成绩单使用是 R 软件环境的一部分热图包28不同库中的每个基因数目计算热图。暗的阴影代表更高的基因表达。1,256 基因的表达显著变化 (Q 值 &#60; 0.05) 显示。基因排序按照他们平均的表达水平，由树状图显示在左边 （而不是根据其亲缘关系） 表示。注意到大量的氨基酸 (AA) 与 20-羟基蜕皮酮 (20E) 刺激后向上或向下调控表达的基因。AP =白纹伊蚊生理盐水。基因和他们相对表达水平的列表，请参见补充文件 1 。<a href="//ecsource.jove.com/files/ftp_upload/55508/55508fig1large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>氨基酸</td>
			<td>相对分子质量 g/mol</td>
			<td>mM 浓度</td>
			<td>毫克 / 升</td>
			<td>300 毫升体积毫克</td>
		</tr>
		<tr>
			<td>丙氨酸</td>
			<td>89.1</td>
			<td>26.68</td>
			<td>2377.19</td>
			<td>713.16</td>
		</tr>
		<tr>
			<td>精氨酸</td>
			<td>174.2</td>
			<td>26.68</td>
			<td>4647.66</td>
			<td>1394.3</td>
		</tr>
		<tr>
			<td>天冬酰胺</td>
			<td>保单增加 150.1</td>
			<td>26.68</td>
			<td>4004.67</td>
			<td>1201.4</td>
		</tr>
		<tr>
			<td>天冬氨酸</td>
			<td>133</td>
			<td>26.68</td>
			<td>3548.44</td>
			<td>1064.53</td>
		</tr>
		<tr>
			<td>半胱氨酸</td>
			<td>121.16</td>
			<td>10.68</td>
			<td>1293.99</td>
			<td>388.2</td>
		</tr>
		<tr>
			<td>谷氨酸</td>
			<td>147.1</td>
			<td>26.68</td>
			<td>3924.63</td>
			<td>1177.39</td>
		</tr>
		<tr>
			<td>谷氨酰胺</td>
			<td>146</td>
			<td>26.68</td>
			<td>3895.28</td>
			<td>1168.58</td>
		</tr>
		<tr>
			<td>甘氨酸</td>
			<td>75</td>
			<td>53.32</td>
			<td>3999</td>
			<td>1199.7</td>
		</tr>
		<tr>
			<td>组氨酸</td>
			<td>155.16</td>
			<td>80</td>
			<td>12412.8</td>
			<td>3723.84</td>
		</tr>
		<tr>
			<td>异亮氨酸</td>
			<td>131</td>
			<td>10.68</td>
			<td>1399.08</td>
			<td>419.72</td>
		</tr>
		<tr>
			<td>甜菜碱</td>
			<td>183</td>
			<td>26.68</td>
			<td>4882.44</td>
			<td>1464.73</td>
		</tr>
		<tr>
			<td>亮氨酸</td>
			<td>131</td>
			<td>26.68</td>
			<td>3495.08</td>
			<td>1048.52</td>
		</tr>
		<tr>
			<td>苯丙氨酸</td>
			<td>165</td>
			<td>10.68</td>
			<td>1762.2</td>
			<td>528.66</td>
		</tr>
		<tr>
			<td>脯氨酸</td>
			<td>115</td>
			<td>26.68</td>
			<td>3068.2</td>
			<td>920.46</td>
		</tr>
		<tr>
			<td>丝氨酸</td>
			<td>105</td>
			<td>53.32</td>
			<td>5598.6</td>
			<td>1679.58</td>
		</tr>
		<tr>
			<td>苏氨酸</td>
			<td>119</td>
			<td>10.68</td>
			<td>1270.92</td>
			<td>381.28</td>
		</tr>
		<tr>
			<td>色氨酸</td>
			<td>204</td>
			<td>10.68</td>
			<td>2178.72</td>
			<td>653.62</td>
		</tr>
		<tr>
			<td>酪氨酸</td>
			<td>181</td>
			<td>5.32</td>
			<td>962.92</td>
			<td>288.88</td>
		</tr>
		<tr>
			<td>缬氨酸</td>
			<td>117</td>
			<td>10.68</td>
			<td>1249.56</td>
			<td>374.87</td>
		</tr>
		<tr>
			<td>蛋氨酸</td>
			<td>149</td>
			<td>10.68</td>
			<td>1591.32</td>
			<td>477.4</td>
		</tr>
	</tbody>
</table>
表 1。4 x 氨基酸原液。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>组件</td>
			<td>在添加到 50 毫升 ddH2O 克重量</td>
		</tr>
		<tr>
			<td>氯化钠</td>
			<td>8.0 g</td>
		</tr>
		<tr>
			<td>氯化钾</td>
			<td>0.074 g</td>
		</tr>
		<tr>
			<td>氯化镁2-6 H2O</td>
			<td>0.120 g</td>
		</tr>
		<tr>
			<td>碳酸氢钠3</td>
			<td>0.0250 g</td>
		</tr>
	</tbody>
</table>
表 2.20 X 盐股票解决方案。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>组件</td>
			<td>重量在克加入 100 毫升 ddH2O</td>
		</tr>
		<tr>
			<td>CaCl2-2 H2O</td>
			<td>0.90 g</td>
		</tr>
	</tbody>
</table>
<stron></stron>g > 表 3。50 x 钙股票解决方案。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>组件</td>
			<td>股票的溶液的浓度</td>
			<td>蓄积量为 100 毫升缓冲区的</td>
		</tr>
		<tr>
			<td>三 pH8.0</td>
			<td>1 米</td>
			<td>5 毫升</td>
		</tr>
		<tr>
			<td>乙二胺四乙酸</td>
			<td>0.25 M</td>
			<td>2 毫升</td>
		</tr>
		<tr>
			<td>氯化钠</td>
			<td>NA</td>
			<td>0.3 g</td>
		</tr>
		<tr>
			<td>ddH2O</td>
			<td>NA</td>
			<td>到 100 毫升 (mL ~ 93)</td>
		</tr>
	</tbody>
</table>
表 4.三缓冲区。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>组件</td>
			<td>蓄积量为 200 毫升</td>
		</tr>
		<tr>
			<td>氨基酸原液</td>
			<td>150 毫升</td>
		</tr>
		<tr>
			<td>盐原液</td>
			<td>10 毫升</td>
		</tr>
		<tr>
			<td>钙股票解决方案</td>
			<td>4 毫升</td>
		</tr>
		<tr>
			<td>工商业污水附加费缓冲区</td>
			<td>10 毫升</td>
		</tr>
		<tr>
			<td>ddH2O</td>
			<td>26 毫升</td>
		</tr>
	</tbody>
</table>
表 5.脂肪体培养基。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>注释</td>
			<td>基因描述</td>
			<td>褶皱变化</td>
			<td>P 值</td>
		</tr>
		<tr>
			<td>AAEL006138</td>
			<td>卵黄蛋白原 B</td>
			<td>3443</td>
			<td>2.52E-112</td>
		</tr>
		<tr>
			<td>AAEL006126</td>
			<td>卵黄蛋白原 C</td>
			<td>2795</td>
			<td>8.64E-91</td>
		</tr>
		<tr>
			<td>AAEL006563</td>
			<td>卵黄羧肽酶</td>
			<td>1002</td>
			<td>2.17E-119</td>
		</tr>
		<tr>
			<td>AAEL010434</td>
			<td>卵黄蛋白原 A</td>
			<td>220</td>
			<td>1.14E-27</td>
		</tr>
		<tr>
			<td>AAEL006542</td>
			<td>卵黄羧肽酶</td>
			<td>185</td>
			<td>2.14E-65</td>
		</tr>
		<tr>
			<td>AAEL012678</td>
			<td>AAEL003006-PA [白纹伊蚊 aegypti](65%)</td>
			<td>96</td>
			<td>4.00E-70</td>
		</tr>
		<tr>
			<td>AAEL000080</td>
			<td>假设蛋白</td>
			<td>82</td>
			<td>6.69E-188</td>
		</tr>
		<tr>
			<td>AAEL015312</td>
			<td>卵黄组织蛋白酶 B</td>
			<td>77</td>
			<td>1.27E-15</td>
		</tr>
		<tr>
			<td>AAEL009588</td>
			<td>核受体 3</td>
			<td>75</td>
			<td>4.58E-56</td>
		</tr>
		<tr>
			<td>AAEL010529</td>
			<td>假设蛋白</td>
			<td>66</td>
			<td>1.32E-29</td>
		</tr>
	</tbody>
</table>
表 6.实验的结果。
补充文件 1。<a href="//ecsource.jove.com/files/ftp_upload/55508/Table_of_genes_for_figure_1.xlsx" target="_blank">请点击这里下载此文件。</a>

Watch this Scientific Journal Video about Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus at JoVE.com

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

脂肪体是昆虫中央代谢器官。我们目前活体器官的文化系统，使用户能够研究分离的脂肪体组织对各种刺激的反应。

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 ...

fat-body-organ-culture-system-in-aedes-aegypti-a-vector-of-zika-virus

Research

JoVE Journal

Biology

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

9.4K 次观看.  New Mexico State University. 脂肪体是昆虫中央代谢器官。我们目前活体器官的文化系统，使用户能够研究分离的脂肪体组织对各种刺激的反应。

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

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

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

原代培养及质粒电穿孔尔蒂小鼠器官。

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

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

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. 
This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis1. 
The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.

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

In this protocol we combine RNAi-mediated gene silencing with an in vivo diuresis assay to study the effects knockdown of genes of interest has on mosquito fluid excretion.

RNAi介导的基因敲除和<em&gt;在体内</em在成年女性利尿含量<em&gt;埃及伊蚊</em&gt;蚊子

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion ...

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of these disease vectors. We have recently identified a bitter sensing neuron in the labellum of female Aedes aegypti that responds to DEET and other repellents, as well as bitter quinine, through direct electrophysiological investigation. These gustatory receptor neuron responses prompted our sequencing of total mRNA from both male and female labella and tarsi samples to elucidate the putative chemoreception genes expressed in these contact chemoreception tissues. Samples of tarsi were divided into pro-, meso- and metathoracic subtypes for both sexes. We then validated our dataset by conducting qRT-PCR on the same tissue samples and used statistical methods to compare results between the two methods. Studies addressing molecular function may now target specific genes to determine those involved in repellent perception by mosquitoes. These receptor pathways may be used to screen novel repellents towards disruption of host-seeking behavior to curb the spread of harmful viruses.

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Using two methods to estimate gene expression in the major gustatory appendages of Aedes aegypti, we have identified the set of genes putatively underlying the neuronal responses to bitter and repulsive compounds, as determined by electrophysiological examination.

生理录音和黄热病蚊子的味觉附属物RNA测序<em&gt;埃及伊蚊</em

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of ...

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 ...

Preparing Irradiated and Marked Male Aedes aegypti Mosquitoes for Release in an Operational Sterile Insect Technique Program

The control of such human diseases as dengue, Zika, and chikungunya relies on the control of their vector, the Aedes aegypti mosquito, because there is no prevention. Control of mosquito vectors can rely on chemicals applied to the immature and adult stages, which can contribute to the mortality of non-targets and more importantly, lead to insecticide resistance in the vector. The sterile insect technique (SIT) is a method of controlling populations of pests through the release of sterilized adult males that mate with wild females to produce non-viable offspring. This paper describes the process of producing sterile males for use in an operational SIT program for the control of Aedes aegypti mosquitoes. Outlined here are the steps used in the program including rearing and maintaining a colony, separating male and female pupae, irradiating and marking adult males, and shipping Aedes aegypti males to the release site. Also discussed are procedural caveats, program limitations, and future objectives.

Preparing Irradiated and Marked Male Aedes aegypti Mosquitoes for Release in an Operational Sterile Insect Technique Program

The sterile insect technique (SIT) is used to control specific, medically important mosquito populations that may be resistant to chemical controls. Here, we describe a method of mass rearing and preparation of sterile male mosquitoes for release in an operational SIT program targeting the Aedes aegypti mosquito.

准备辐照和标记的雄性 埃及伊蚊 ，以便在操作性昆虫技术计划中释放

The control of such human diseases as dengue, Zika, and chikungunya relies on the control of their vector, the Aedes aegypti mosquito, because there is no ...

Using the Fluorescent Dye, Rhodamine B, to Study Mating Competitiveness in Male Aedes aegypti Mosquitoes

The success of sterile or incompatible insect technique-based population suppression programs depends on the ability of released males to compete for wild-type females and induce sterility in the target population. Hence, laboratory assessment of male mating competitiveness is essential for evaluating the release strain&#8217;s fitness before field release. Conventionally, such an assay is performed by determining the proportion of viable eggs produced by the females after being simultaneously exposed to two sets of males (wild-type and release strains) for copulation. However, this process is time-consuming and laborious due to the need to first blood-feed the females for egg production and then hatch and enumerate the hatched eggs to determine egg viability.&#160;
Moreover, this method cannot discern the degree of competitiveness between two sterile or Wolbachia-infected mosquito lines as wild-type female mosquitoes will only produce non-viable eggs upon mating with both. To circumvent these limitations, this paper describes a more direct method of measuring male mosquito mating competitiveness in laboratory settings using the fluorescent dye, rhodamine B (RhB), which can be used to mark males by feeding them in sucrose solution containing RhB. After the mating assay, the presence of fluorescing sperms in the spermathecae of a female can be used to determine her mating partner. This method is cost-effective, reduces the experimental time by 90% and allows comparison of mating fitness between two sterile or Wolbachia-infected lines.&#160;

Using the Fluorescent Dye, Rhodamine B, to Study Mating Competitiveness in Male Aedes aegypti Mosquitoes

Here, we present a protocol for studying mating competitiveness of male Aedes aegypti using fluorescent dye as a marker. Female mosquitoes are exposed to both marked and unmarked males for copulation. Post mating, their spermathecae are examined under a fluorescence microscope to determine their mating partner.

使用荧光染料，罗达明B，研究雄性 伊蚊 的交配竞争力

The success of sterile or incompatible insect technique-based population suppression programs depends on the ability of released males to compete for ...

Immunofluorescent Labeling of Plant Virus and Insect Vector Proteins in Hemipteran Guts

Most plant viruses in nature are transmitted from one plant to another by hemipteran insects. A high population density of the vector insects that are highly efficient at virus transmission plays a key role in virus epidemics in fields. Studying virus-insect vector interactions can advance our understanding of virus transmission and epidemics with the aim of designing novel strategies to control plant viruses and their vector insects. Immunofluorescence labeling has been widely used to analyze interactions between pathogens and hosts and is used here in the white-backed planthopper (WBPH, Sogatella furcifera), which efficiently transmits the southern rice black streaked dwarf virus (SRBSDV, genus Fijivirus, family Reoviridae), to locate the virions and insect proteins in the midgut epithelial cells. Using laser scanning confocal microscopy, we studied the morphological characteristics of midgut epithelial cells, cellular localization of insect proteins, and the colocalization of virions and an insect protein. This protocol can be used to study virus activities in insects, functions of insect proteins, and interactions between virus and vector insect.

This protocol for immunofluorescent labeling of both plant virus proteins and vector insect proteins in excised insect guts can be used to study interactions among virus and vector insects, insect protein functions and molecular mechanisms underlying virus transmission.

半翅目肠道中植物病毒和昆虫载体蛋白的免疫荧光标记

Most plant viruses in nature are transmitted from one plant to another by hemipteran insects. A high population density of the vector insects that are ...

Process Development for the Production and Purification of Adeno-Associated Virus (AAV)2 Vector using Baculovirus-Insect Cell Culture System

Adeno-associated viruses (AAV) are promising vectors for gene therapy applications. Here, the AAV2 vector is produced by co-culture of Spodoptera frugiperda (Sf9) cells with Sf9 cells infected with baculovirus (BV)-AAV2-GFP (or therapeutic gene) and BV-AAV2-rep-cap in serum-free suspension culture. Cells are cultured in a flask in an orbital shaker or Wave bioreactor. To release the AAV particles, producer cells are lysed in buffer containing detergent, which is subsequently clarified by low-speed centrifugation and filtration. AAV particles are purified from the cell lysate using AVB Sepharose column chromatography, which binds AAV particles. Bound particles are washed with PBS to remove contaminants and eluted from the resin using sodium citrate buffer at pH 3.0. The acidic eluate is neutralized with alkaline Tris-HCl buffer (pH 8.0), diluted with phosphate-buffered saline (PBS), and further concentrated with tangential flow filtration (TFF). The protocol describes small-scale pre-clinical vector production compatible with scale-up to large-scale clinical-grade AAV manufacturing for human gene therapy applications.

Process Development for the Production and Purification of Adeno-Associated Virus AAV2 Vector using Baculovirus-Insect Cell Culture System

In this protocol, AAV2 vector is produced by co-culturing Spodoptera frugiperda (Sf9) insect cells with baculovirus (BV)-AAV2-green fluorescent protein (GFP) or therapeutic gene and BV-AAV2-rep-cap infected Sf9 cells in suspension culture. AAV particles are released from the cells using detergent, clarified, purified by affinity column chromatography, and concentrated by tangential flow filtration.

肥胖的身体器官培养体系中蚊，一个向量 Zika 病毒

摘要

探索更多视频

此视频中的章节

女答：埃及伊蚊蚊的dsRNA注入

原代培养及质粒电穿孔尔蒂小鼠器官。

RNAi介导的基因敲除和

生理录音和黄热病蚊子的味觉附属物RNA测序

维护伊蚊蚊蚊子感染沃尔巴克

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

准备辐照和标记的雄性埃及伊蚊，以便在操作性昆虫技术计划中释放

使用荧光染料，罗达明B，研究雄性伊蚊的交配竞争力

半翅目肠道中植物病毒和昆虫载体蛋白的免疫荧光标记

使用杆状病毒-昆虫细胞培养系统生产和纯化腺相关病毒（AAV）2载体的工艺开发

肥胖的身体器官培养体系中蚊，一个向量 Zika 病毒

摘要

探索更多视频

此视频中的章节

女答：埃及伊蚊蚊的dsRNA注入

原代培养及质粒电穿孔尔蒂小鼠器官。

RNAi介导的基因敲除和

生理录音和黄热病蚊子的味觉附属物RNA测序

维护伊蚊蚊蚊子感染沃尔巴克

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

准备辐照和标记的雄性 埃及伊蚊 ，以便在操作性昆虫技术计划中释放

使用荧光染料，罗达明B，研究雄性 伊蚊 的交配竞争力

半翅目肠道中植物病毒和昆虫载体蛋白的免疫荧光标记

使用杆状病毒-昆虫细胞培养系统生产和纯化腺相关病毒（AAV）2载体的工艺开发

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

准备辐照和标记的雄性埃及伊蚊，以便在操作性昆虫技术计划中释放

使用荧光染料，罗达明B，研究雄性伊蚊的交配竞争力