Name: fat body organ culture system in aedes aegypti, a vector of zika virus
Uploaded: 2017-08-19T13:00:00.000+00:00
Duration: 6 min 18 s
Description: Watch this Scientific Journal Video about Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus at JoVE.com

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>

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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><tbody><tr><td>Scissors&amp;nbsp;</td><td>Fiskars</td><td>83872</td><td></td></tr><tr><td>Fly pad</td><td>Genesee Scientific</td><td>789060</td><td></td></tr><tr><td>Battery-powered aspirator w/ collection vial</td><td>Hausherrs Machine Works, Inc.</td><td>3740-01-210-2368</td><td></td></tr><tr><td>Fine tip forceps</td><td>World Precision Instruments, Inc.&amp;nbsp;</td><td>500085</td><td></td></tr><tr><td>Light microscope&amp;nbsp;</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>96 well plate&amp;nbsp;</td><td>Sigma</td><td>CL S3383</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S9378</td><td></td></tr><tr><td>Alanine</td><td>Sigma</td><td>A7627</td><td></td></tr><tr><td>Arginine</td><td>Sigma</td><td>A5006</td><td></td></tr><tr><td>Asparagine</td><td>Sigma</td><td>A0884</td><td></td></tr><tr><td>Aspartic Acid</td><td>Sigma</td><td>A9256</td><td></td></tr><tr><td>Cysteine</td><td>Sigma</td><td>W326305</td><td></td></tr><tr><td>Glutamic Acid</td><td>Sigma</td><td>G1251</td><td></td></tr><tr><td>Glutamine</td><td>Sigma</td><td>G3126</td><td></td></tr><tr><td>Glycine</td><td>Sigma</td><td>G2879</td><td></td></tr><tr><td>Histidine</td><td>Sigma</td><td>H6034</td><td></td></tr><tr><td>Isoleucine</td><td>Sigma</td><td>I2752</td><td></td></tr><tr><td>Lysine</td><td>Sigma</td><td>L5501</td><td></td></tr><tr><td>Leucine</td><td>Sigma</td><td>L8000</td><td></td></tr><tr><td>Phenylalanine</td><td>Sigma</td><td>P2126</td><td></td></tr><tr><td>Proline</td><td>Sigma</td><td>P0380</td><td></td></tr><tr><td>Serine</td><td>Sigma</td><td>S4500</td><td></td></tr><tr><td>Threonine</td><td>Sigma</td><td>T8625</td><td></td></tr><tr><td>Tryptophan</td><td>Sigma</td><td>T0254</td><td></td></tr><tr><td>Tyrosine</td><td>Sigma</td><td>T3754</td><td></td></tr><tr><td>Valine</td><td>Sigma</td><td>V0500</td><td></td></tr><tr><td>Methionine</td><td>Sigma</td><td>M9625</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S7653</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P9333</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;-6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>M2670</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S5761</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;-2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>C8106</td><td></td></tr><tr><td>Tris pH8.0&amp;nbsp;</td><td>Sigma</td><td>T1503</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>E6758</td><td></td></tr><tr><td>ddH&lt;sub&gt;2&lt;/sub&gt;O</td><td>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 ...

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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

Alphavirus Transducing System:  Tools for Visualizing Infection in Mosquito Vectors

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection. ATSs are derived from cDNA clones of mosquito-borne RNA viruses (genus Alphavirus; family Togaviridae). The Alphavirus genus contains about 30 different mosquito-borne virus species. Alphaviruses are enveloped viruses and contain single-stranded RNA genomes (~11.7 Kb). Alphaviruses transcribe a subgenomic mRNA that encodes the structural proteins of the virus required for encapsidation of the genome and maturation of the virus. Alphaviruses are usually highly lytic in vertebrate cells, but persistently infect susceptible mosquito cells with minimal cytopathology. These attributes make them excellent tools for gene expression in mosquito vectors. The most common ATSs in use are derived from Sindbis virus (SINV). The broad species tropism of SINV allows for infection of insect, avian, and mammalian cells8. However, ATSs have been derived from other alphaviruses as well9,10,20. Foreign gene expression is made possible by the insertion of an additional viral subgenomic RNA initiation site or promoter. ATSs in which an exogenous gene sequence is positioned 5' to the viral structural genes is used for stable protein expression in insects. ATSs, in which a gene sequence is positioned 3' to the structural genes, is used to trigger RNAi and silence expression of that gene in the insect.
ATSs have proven to be valuable tools for understanding vector-pathogen interactions, molecular details of viral replication and maintenance infectious cycles3,4,11,19,21. In particular, the expression of fluorescent and bioluminescent reporters has been instrumental tracking the viral infection in the vector and virus transmission5,14-16,18. Additionally, the vector immune response has been described using two strains of SINV engineered to express GFP2,9.
Here, we present a method for the production of SINV containing a fluorescent reporter (GFP) from the cDNA infectious clone. Infectious, full-length RNA is transcribed from the linearized cDNA clone. Infectious RNA is introduced into permissive target cells by electroporation. Transfected cells generate infectious virus particles expressing the GOI. Harvested virus is used to infect mosquitoes, as described here, or other host species (not shown herein). Vector competence is assessed by detecting fluorescence outside the midgut or by monitoring virus transmission7. Use of a fluorescent reporter as the GOI allows for convenient estimation of virus spread throughout a cell culture, for determination of rate of infection, dissemination in exposed mosquitoes, virus transmission from the mosquito and provides a rapid gauge of vector competence.

Methods for using alphavirus transducing systems to express fluorescent reporters in vitro and in adult mosquitoes are described. This technique may be adapted to express any protein of interest in lieu of or in addition to a reporter.

甲病毒转导系统：可视化，以免蚊虫感染的工具

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection.  ATSs are derived from cDNA clones of ...

科研

免疫与感染

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 ...

生物学

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV is an RNA virus of the Flaviviridae family. ZIKV is mainly transmitted by mosquitoes, but can also be spread by maternal to fetal vertical transmission as well as sexual contact. To date, there are no reliable treatment or vaccine options available to protect those infected by the virus. The development of a reproducible, effective Zika virus infectious cell culture system is critical for studying the molecular mechanisms of ZIKV replication as well as drug and vaccine development. In this regard, a protocol describing a mammalian cell-based in vitro Zika virus culture system for viral production and growth analysis is reported here. Details on the formation of plaques by Zika virus on a cell monolayer and plaque assay for measuring viral titer are presented. Viral genome replication kinetics and double-stranded RNA genome replicatory intermediates are determined. This culture platform was utilized to screen against a library of a small set of cytokines resulting in the identification of interferon-&#945; (IFN-&#945;), IFN-&#946; and IFN-&#947; as potent inhibitors of Zika viral growth. In summary, an in vitro infectious Zika viral culture system and various virological assays are demonstrated in this study, which has the potential to greatly benefit the research community in elucidating further the mechanisms of viral pathogenesis and the evolution of viral virulence. Antiviral IFN-alpha can further be evaluated as a prophylactic, post-exposure prophylactic, and treatment option for Zika virus infections in high-risk populations, including infected pregnant women.

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV), an emerging pathogen, is linked to fetal developmental abnormalities and microcephaly. The establishment of an effective infectious cell culture system is crucial for studies of ZIKV replication as well as vaccine and drug development. In this study, various virological assays pertaining to ZIKV are illustrated and discussed.

兹卡病毒感染细胞培养系统和<em&gt;体外</em&gt;干扰素的预防作用

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV ...

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine the rate of infection, disseminated infection, and potential transmission of the virus in the mosquito population in question. These procedures are widely utilized with various modifications in vector competence evaluations globally. They are important in determining the potential role that a given mosquito (i.e., species, population, individual) may play in the transmission of a given agent.

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The presented protocol can determine the vector competence of Aedes aegypti mosquito populations for a given virus, such as Zika, in a containment setting.

使用寨卡病毒对 埃及伊 蚊的载体能力分析

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine ...

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of disease cases each year. E. aedis causes mortality and reduced reproductive fitness in the mosquito vector and has been explored for its potential as a biocontrol agent. The protocol we present for culturing E. aedis is based on its natural infection cycle, which involves both horizontal and vertical transmission at different life stages of the mosquito host. Ae. aegypti mosquitoes are exposed to spores in the larval stage. These infected larvae then mature into adults and transmit the parasite vertically to their offspring. Infected offspring are then used as a source of spores for future horizontal transmission. Culturing E. aedis can be challenging to the uninitiated given the complexities of the parasite&#8217;s life cycle, and this protocol provides detailed guidance and visual aids for clarification.

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

A protocol to culture the microsporidian parasite Edhazardia aedis. The parasite is passaged from one generation of Aedes aegypti mosquitoes to the next via horizontal transfer at the larval stage followed by vertical transmission at the adult stage. Live sporoplasms survive long-term in infected eggs.

在伊蚊中传播微孢子寄生虫Edhazardia aedis

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of ...

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In the laboratory, researchers can deliver animal-derived and artificial blood meals to mosquitoes via membrane feeders, which allow for manipulation of meal composition. Here, we present methods for feeding blood and artificial blood meals to Aedes aegypti mosquitoes and quantifying the volume consumed by individual females.
Targeted feeding and quantification of artificial/blood meals have broad uses, including testing the effects of meal components on mosquito behavior and physiology, delivering pharmacological compounds without injection, and infecting mosquitoes with specific pathogens. Adding fluorescein dye to the meal prior to feeding allows for subsequent meal size quantification. The meal volume consumed by mosquitoes can be measured either by weight, if the females are to be used later for behavioral experiments, or by homogenizing individual females in 96-well plates and measuring fluorescence levels using a plate reader as an endpoint assay. Meal size quantification can be used to determine whether changing the meal components alters the meal volume ingested or if meal consumption differs between mosquito strains. Precise meal size quantification is also critical for downstream assays, such as those measuring effects on host attraction or fecundity. The methods presented here can be further adapted to track meal digestion over the course of days or to include multiple distinguishable markers added to different meals (like nectar and blood) to quantify the consumption of each meal by a single mosquito.
These methods allow researchers to singlehandedly perform high-throughput measurements to compare the meal volume consumed by hundreds of individual mosquitoes. These tools will therefore be broadly useful to the community of mosquito researchers for answering diverse biological questions.

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

The goal of this protocol is to deliver animal-derived and artificial blood meals to Aedes aegypti mosquitoes through an artificial membrane feeder and precisely quantify the volume of meal ingested.

在 伊 蚊中喂养和量化动物衍生的血液和人工膳食

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In ...

Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection

Mosquito-borne viruses (MBVs), which are infectious pathogens to vertebrates, are spread by many mosquito species, posing a severe threat to public health. Once ingested, the viruses must overcome the mosquito midgut barrier to reach the hemolymph, from where they might potentially spread to the salivary glands. When a mosquito bites, these viruses are spread to new vertebrate hosts. Similarly, the mosquito may pick up different viruses. In general, only a tiny portion of viruses may enter the salivary glands via the gut. The transmission efficiency of these viruses to the glands is be affected by the two physical barriers found in different mosquito species: midgut barriers and salivary glands barriers. This protocol presents a method for virus detection in salivary glands of Aedes aegypti&#39;s&#160;following oral feeding and intrathoracic injection infection. Furthermore, determining whether the guts and/or salivary glands hinder viral spread can aid in the risk assessments of MBVs transmitted by Aedes aegypti.

This methodology, which included oral feeding and intrathoracic injection infection, could effectively assess the influence of midgut and/or salivary gland barriers on arbovirus infection.

经口喂养和显微注射对成年蚊子进行实验性病毒感染

Mosquito-borne viruses (MBVs), which are infectious pathogens to vertebrates, are spread by many mosquito species, posing a severe threat to public ...

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, ...

Forced Salivation As a Method to Analyze Vector Competence of Mosquitoes

Vector competence is defined as the potential of a mosquito species to transmit a mosquito-borne virus (mobovirus) to a vertebrate host. Viable virus particles are transmitted during a blood meal via the saliva of an infected mosquito. Forced salivation assays allow determining the vector potential on the basis of single mosquitoes, avoiding the use of animal experiments. The method is suitable to analyze a large number of mosquitoes in one experiment within a short period of time. Forced salivation assays were used to analyze 856 individual mosquitoes trapped in Germany, including two different Culex pipiens pipiens biotypes, Culex torrentium as well as Aedes albopictus, which were experimentally infected with Zika virus (ZIKV) and incubated at 18 &#176;C or 27 &#176;C for two and three weeks. The results indicated the lack of vector competence of the different Culex taxa for ZIKV. In contrast, Aedes albopictus was susceptible to ZIKV, but only at 27 &#176;C, with transmission rates similar to an Aedes aegypti laboratory colony tested in parallel.

For efficient control of mosquito borne virus transmission, the knowledge of the vector potential of respective mosquitoes is of particular interest. We describe forced salivation as a method to analyze vector competence of Aedes albopictus and three different Culex taxa for the transmission of Zika virus.

强迫流涎法分析蚊虫的载体能力

Vector competence is defined as the potential of a mosquito species to transmit a mosquito-borne virus (mobovirus) to a vertebrate host. Viable virus ...

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 ...

肥胖的身体器官培养体系中蚊，一个向量 Zika 病毒

摘要

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此视频中的章节

甲病毒转导系统：可视化，以免蚊虫感染的工具

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“

兹卡病毒感染细胞培养系统和

使用寨卡病毒对埃及伊蚊的载体能力分析

在伊蚊中传播微孢子寄生虫Edhazardia aedis

在伊蚊中喂养和量化动物衍生的血液和人工膳食

经口喂养和显微注射对成年蚊子进行实验性病毒感染

维护伊蚊蚊蚊子感染沃尔巴克

强迫流涎法分析蚊虫的载体能力

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

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

甲病毒转导系统：可视化，以免蚊虫感染的工具

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“

兹卡病毒感染细胞培养系统和

使用寨卡病毒对 埃及伊 蚊的载体能力分析

在伊蚊中传播微孢子寄生虫Edhazardia aedis

在 伊 蚊中喂养和量化动物衍生的血液和人工膳食

经口喂养和显微注射对成年蚊子进行实验性病毒感染

维护伊蚊蚊蚊子感染沃尔巴克

强迫流涎法分析蚊虫的载体能力

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

使用寨卡病毒对埃及伊蚊的载体能力分析

在伊蚊中喂养和量化动物衍生的血液和人工膳食

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