我们感谢艾拉 Weinert, 米歇尔赫尔姆斯和 Marlis Badusche 为优秀的技术援助, 杰西卡 Börstler 分析淡色蚊子, 罗伯特贝克尔和 Björn Pluskota 提供白纹伊蚊蛋和为 Vapalahti提供病毒储存。ML 得到了莱布尼茨协会的支持;授予编号 SAW-2014-SGN-3。

1. 准备雌性蚊子
<ol><li>使用一个吸引器 (大约400蚊子每大笼子) 收集蚊子从一个大笼子。</li>
	<li>麻醉蚊子与二氧化碳 (CO2) 为 7 s。</li>
	<li>每塑料瓶20位女性 (Ø50毫米 x 100 毫米)。 注: 如果蚊子醒了, 使用另外 3 s CO2.
</li>
	<li>一夜之间饿死蚊子。</li>
</ol>2. 人工血粉
注意: 所有步骤都在 BSL-3 条件下执行。
<ol><li>
准备传染性的血餐。
	<ol>
<li>将病毒储存稀释至 1 x 108斑块成型单元 (PFU)/毫升浓度。 注意: 通过执行组织培养感染剂量50方法 (TCID50), 并由长矛 & Kärber 算法10,11计算出库存浓度。</li>
		<li>混合过期的人血 (血液保存) 从血库 (不适合人类, 但有用的蚊子) 与果糖 (8% 溶液), 过滤牛血清 (血清) 和工作溶液的病毒储存 (过期的人类 blood:fructose:FBS:工作解决方案 = 5:3: 1:1)。 注: 我们选择了人类的血液, 因为这是已知的天然哺乳动物宿主的 ZIKV。</li>
	</ol>
</li>
	<li>冷冻140µL 的血粉混合供进一步分析通过 TCID50。</li>
	<li>
使用不同的喂养方法进行人工喂养, 具体取决于种类:
	<ol>
<li>
白纹蚊: 使用膜喂养系统30分钟 (每馈线1毫升)。</li>
		<li>
淡色: 一夜之间通过棉花棒提供传染性血液, 不加热血液 (每瓶300µL)。</li>
		<li>
白纹伊蚊: 把两滴 (50 µL) 的血餐混合到塑料瓶中, 让蚊子喂养2小时。不要加热血液。 注意: 蚊子会在头15分钟内进食。由于安全原因, 长时间的潜伏期是必要的。血滴在2小时后排出, 因此在喂食蚊子的分类过程中减少了污染风险。</li>
	</ol>
</li>
	<li>麻醉蚊子与 CO2为 7 s。 注: 如果蚊子醒了, 使用另外 3 s CO2。</li>
	<li>排序和计数完全肿胀蚊子进入一个新的瓶子。</li>
	<li>添加一个棉垫, 饱和与果糖 (8% 溶液) 之间的瓶子和插头。</li>
	<li>保持蚊子在指定的温度18°c 或27°c 在湿气80% 为14或21天。</li>
	<li>用果糖饱和的棉垫喂蚊子。每瓶72小时补充1.5 毫升果糖 (8% 溶液) (最多20只蚊子, 取决于喂养速率, 见步骤 2.5)。</li>
</ol>3. 14 或21天强迫流涎化验
注意: 所有步骤都在 BSL-3 条件下执行。
<ol><li>种子 2 x 104维罗细胞/每井在96个井板与200µL 生长培养基 (Dulbecco´s 修饰的老鹰培养基 (DMEM) 补充3% 血清, 100 U/毫升青霉素, 100 毫克/毫升链霉素, 5 µg/毫升两性霉素 B, 2 毫米 l-谷氨酰胺, 1% 非必需氨基酸 (NEAA), 1% 丙酮酸钠)。</li>
	<li>
准备流涎设备:
	<ol>
<li>将方形玻璃板 (20 x 20 厘米) 放置在工作台上的一个角度。 注意: 这是必要的, 因为冲击波在安全实验室。如果板材是水平或垂直的, 液体将泄漏。</li>
		<li>将双面胶带放在玻璃板的顶部。</li>
		<li>作用的建模粘土, 直到它达到直径0.5 厘米, 并将其水平1厘米, 远离双面胶带。</li>
		<li>切断10µL 过滤尖端尖端的前0.3 厘米。 注: 建议在生物安全实验室以外的地方开始实验前做好准备。</li>
		<li>用10µL 磷酸盐缓冲盐水 (PBS, pH 值 7.4) 填充过滤器提示。</li>
		<li>将过滤器提示放在建模粘土上, 并向胶带倾斜。</li>
		<li>使用柔和的压力来保护过滤器提示。</li>
	</ol>
</li>
	<li>
准备蚊子:
	<ol>
<li>麻醉蚊子与 CO2。</li>
		<li>去掉腿和翅膀, 使蚊子不动。</li>
		<li>把活的蚊子尸体固定在过滤器尖端上面的粘胶带上。</li>
		<li>将喙轻轻放进过滤器尖端。</li>
		<li>运行流涎化验30分钟。</li>
		<li>删除过滤器提示, 丢弃粘土和胶带。</li>
	</ol>
</li>
</ol>4. 唾液的处理
注意: 在 BSL-3 条件下执行 4.1–4.6 2 步。
<ol><li>将这些内容排出到含有10µL 的反应管中。</li>
	<li>轻轻混合, 并将内容转移到准备好的96井板的水井中, 每样都用一个井。</li>
	<li>孵化板材7天在37°c 与 5% CO2。</li>
	<li>使用双目检查细胞是否存在病变效应 (CPE)。</li>
	<li>如果该井是积极的 CPE, 收获上清的吹打140µL 成一个反应管的 RNA 提取。</li>
	<li>
使用 rna 提取试剂盒纯化 rna。
	<ol>
<li>混合140µL 的上清与560µL 的 AVL 缓冲 (病毒裂解缓冲) 和孵化10分钟室温。</li>
		<li>添加560µL 乙醇 (96%) 和涡流样品。 注: 此步骤后, 样品被灭活, 可以出院的 BSL-3 实验室。</li>
		<li>吸管630µL 的样品到一列和离心机与 6000 x g 为1分钟。</li>
		<li>丢弃滤液, 吸管剩余的630µL 在柱上, 重复离心。</li>
		<li>将500µL 的洗涤缓冲器1加到柱上, 离心机在 6000 x g处进行1分钟, 将 RNA 与柱膜结合。</li>
		<li>丢弃收集管并将该列放入新的收集管中。添加500µL 洗涤缓冲2和离心机在 12500 x g为3分钟。</li>
		<li>丢弃收集管并将该列放入1.5 毫升反应管中。</li>
		<li>添加50µL 洗脱缓冲液, 在室温下孵育1分钟。</li>
		<li>离心机在 6000 x g 1 分钟, 然后丢弃该列。</li>
	</ol>
</li>
	<li>通过 RT-qPCR 分析 ZIKV RNA 的样品。</li>
</ol>5. 蚊子身体的加工
注意: 步骤5.1–5.5 是在 BSL-3 条件下执行的。
<ol><li>除去蚊子的身体, 把每个身体放到一个反应管里。 注意: 此时可以暂停实验, 并将样品存储在摄氏-80 摄氏度。</li>
	<li>在反应管中加入500µL 均匀介质 (DMEM 无任何 supplementals)。</li>
	<li>融汇使用手马达搅拌机, 并为每个样品使用新的雌蕊。</li>
	<li>将匀浆的200µL 加入96井板进行提纯。 注意: 此时可以暂停实验, 并将样品存储在摄氏-80 摄氏度。</li>
	<li>在60摄氏度处孵化60分钟, 使板材失效。</li>
	<li>通过全自动核酸提纯纯化 RNA。 注意: 此时可以暂停实验, 并将样品存储在摄氏-80 摄氏度。</li>
</ol>6. 分析
<ol><li>使用 RT qPCR 量化病毒 RNA 拷贝。 注意: RNA 拷贝是平均的在所有 ZIKV 正面蚊子身体和不包括 ZIKV 阴性蚊子。</li>
	<li>计算 IR, 定义为 ZIKV 阳性蚊子身体的数量每位喂养的女性。</li>
	<li>计算 TR, 定义为每数量 ZIKV 阳性的蚊子身体 ZIKV 阳性唾液的数量。没有 ZIKV 阳性体的物种温度组合不能计算出传输速率。</li>
</ol>

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作者没有什么可透露的。

以前已经表明, 强迫流涎获得的结果与经典的再喂养实验6一致。然而, 无论是在重新喂养实验和我们提出的强迫流涎的方法, 它是不可能直接证明流涎活动或监测唾液释放。为了证明流涎的活性, 样品可以检测唾液中的其他成分 (例如,蛋白质, 碳水化合物等)。此外, 额外的 qPCRs 将允许检测病毒 RNA 拷贝。然而, 这需要在不同的分析中分离唾液样本, 这可能会限制细胞培养实验中极低颗粒数的敏感性。反过来, 这可能导致低估了已确定的传输速率。
为了重现的结果, 有必要确保所有蚊子活着直到实验结束。这是通过视觉监测蚊子的身体运动活动来控制的。此外, 对某种蚊子物种的流涎试验的原则使用必须通过喂养控制病毒进行测试, 已知这类蚊子是由这种特定的蚊虫传播的。反之亦然, 在实验中引入的每种病毒都必须在易感蚊子中进行测试。
强迫流涎测定有许多优点。高数量的蚊子可以同时测试在一个单一的标本基地在控制标准化的条件下, 而不与动物福利条例9的冲突。
该方法由几个实验室使用。然而, 确切的设置可能会有所不同, 这可能导致实验室之间的结果差异。一个关键的组成部分是毛细管收集唾液, 可以由玻璃14或塑料15。为了符合安全等级 3 insectary 的高安全性规定, 我们使用了塑料过滤器的小贴士代替了玻璃毛细血管, 从而降低了受伤的风险。此外, 过滤器提示有优势, 收集的液体可以很容易地转移使用吸管。
这里提出的强制流涎试验的设置是基于两个调查人员一起工作和分担职责。蚊子的复员是由一个人执行的, 另一个则同时准备强制流涎设置。这大大减少了处理时间, 并减少了样本切换的风险, 因为在复员工作场所和流涎之间的变化是不必要的。此外, 它允许在复员后立即将蚊子放在强制流涎装置上。这相当短的蚊子处理时间是必不可少的成功强迫流涎。最后, 在整个实验中可以直接监测蚊子的运动。
本文介绍的流涎分析方法是为了深入了解蚊虫的载体电位。然而, 为了回答其他一些具体问题 (例如, 感染脊椎动物所需的蚊子叮咬数量), 动物实验可能仍然需要。

在 2015年, Zika 病毒 (ZIKV) 是属于家庭Flaviviridae的一种蚊媒病毒 (mobovirus), 出现在哥伦比亚和巴西, 在美洲和加勒比地区迅速蔓延, 引起了大量相关的流行病。小头和 Guillain–Barré综合征1例临床病例。蚊和白纹伊蚊的蚊子被认为是 ZIKV2的主要和次要载体, 但白纹伊属的其他种类也被证明具有实验向量能力3.与伊蚊不同的是, 迄今为止测试的大多数淡色物种都无法传播病毒4。只是,淡色致倦的数据提供了不确定的结果3。目前, 在中等温度条件下 (< 20 摄氏度), ZIKV 的蚊媒能力缺乏信息。然而, 这一信息对于可能蔓延到诸如中欧等温带气候地区的风险评估是很重要的。
ZIKV 的有效载体能够获得、维持、放大并最终传播病毒。因此, 对 ZIKV 的蚊体或身体部位, 包括腿部、中肠、头或涎腺进行检测, 不足以确定载体的能力。在释放的唾液中检测感染病毒微粒是强制性的。为了评估向量的能力, 两种感染率 (IR, 每数量的 ZIKV 阳性蚊子的数量) 和传播率 (TR, 蚊子数量与 ZIKV 阳性唾液每数量 ZIKV 阳性蚊子身体), 必须确定。蚊子的 IRs 可以很容易地通过简单的均质蚊子分析, 其次是反向逆转录酶链反应 (RT qPCR) 靶向 ZIKV。此外, 感染还可以通过确定每只蚊子的病毒拷贝数来进一步表征。相比之下, 传输速率的分析依赖于在个体蚊子唾液中检测出可行的病毒微粒。这可以通过喂养受感染的蚊子在敏感的寄主动物, 其次是分析的病毒血症在主机5。然而, 这种方法依赖于合适的模型有机体, 而且代价高昂, 受到动物福利法规的限制。为了避免使用实验动物来分析传输速率和降低成本, 使用血滴的人工再喂养系统被描述了6。然而, 个体规模的测试与大量个体的结合是困难和耗时的。流涎的第一个描述在个体尺度被出版了在 1966年7年, 并且在近几年, 强迫流涎实验成为了评估蚊子的媒介能力的选择方法3,8.
根据我们的媒介能力研究最近出版的中欧蚊子9, 我们描述强迫流涎报告由安德森et。8与一些修改。该方法允许高生物安全水平条件下的高通量标准化实验 (BSL-3), 包括基于细胞培养的活性病毒的鉴定。这里所描述的实验包括856只蚊子。他们通过人工血餐感染 ZIKV, 随后对唾液中感染性病毒微粒的存在进行分析。在实验中引入的蚊子包括两个长期建立的 Ae 的实验室种群. 蚊和淡色库蚊虱molestus (Cx molestus), 以及田间捕捉淡色库蚊虱库蚊 (cx),淡色 torrentium (cx torrentium) 和两个Ae. 伊蚊种群。除了其中的两个Ae.在意大利收集的伊蚊种群中, 所有蚊子都是在德国收集的。

<table>
	<tbody>
		<tr>
			<td>Inject+matic Sleeper</td>
			<td>Inject+matic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bugdorm-1 Insect Rearing Cage</td>
			<td>BugDorm Store</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Drosophila cultivation tubes</td>
			<td>carl roth</td>
			<td>PK11.2</td>
			<td>plastic vial for mosquitoes</td>
		</tr>
		<tr>
			<td>Plug for drosophila cultivation tubes</td>
			<td>carl roth</td>
			<td>PK15.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Constant climate chamber</td>
			<td>Binder</td>
			<td>KBF-240</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood</td>
			<td></td>
			<td></td>
			<td>banked human blood, expired, not usable for medical application</td>
		</tr>
		<tr>
			<td>Dumont-Pincette Electronic SS</td>
			<td>Dumont</td>
			<td>B40c</td>
			<td></td>
		</tr>
		<tr>
			<td>Vero cells</td>
			<td>BNITM</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flash light aspirator</td>
			<td>Bioquip</td>
			<td>2809</td>
			<td></td>
		</tr>
		<tr>
			<td>double-sided adhesive tape</td>
			<td></td>
			<td></td>
			<td>no specific provider</td>
		</tr>
		<tr>
			<td>Gammex Powder-free gloves with AMT</td>
			<td>Ansell</td>
			<td></td>
			<td>for safety reasons necessary during work with pricks in the BSL3 laboratory</td>
		</tr>
		<tr>
			<td>RealStar Zika Virus RT-PCR-Kit</td>
			<td>altona diagnostics</td>
			<td>591013</td>
			<td></td>
		</tr>
		<tr>
			<td>MagMax Pathogen RNA/DNA Kit</td>
			<td>Thermo fisher scientific</td>
			<td>4462359</td>
			<td>RNA isolation of mosquito bodies</td>
		</tr>
		<tr>
			<td>Qiamp viral RNA Mini Kit</td>
			<td>Qiagen</td>
			<td>52906</td>
			<td>RNA-Isolation of supernatant</td>
		</tr>
		<tr>
			<td>filter tips (20 &#181;L)</td>
			<td>Sarstedt</td>
			<td>701,116,210</td>
			<td>Cut the first 3 mm of the tip</td>
		</tr>
		<tr>
			<td>hand motor mixer</td>
			<td>carl roth</td>
			<td>sold out</td>
			<td></td>
		</tr>
		<tr>
			<td>micro pestles for hand motor mixer</td>
			<td>carl roth</td>
			<td>CXH8.1</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td></td>
			<td>P0403550</td>
			<td></td>
		</tr>
		<tr>
			<td>L Glutamine Penicilin/Streptomycin</td>
			<td>PAN</td>
			<td>P0819100</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>PAN</td>
			<td>P06-0110</td>
			<td></td>
		</tr>
		<tr>
			<td>Fructose</td>
			<td>carl roth</td>
			<td>4981.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>PAN</td>
			<td>P04-36500</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>PAN</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvat</td>
			<td>PAN</td>
			<td>P0443100</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM NAA</td>
			<td>PAN</td>
			<td>P0832100</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemotek Feeder</td>
			<td>Hemotek</td>
			<td>PS6</td>
			<td></td>
		</tr>
		<tr>
			<td>Light Cycler</td>
			<td>Roche</td>
			<td>480 II</td>
			<td></td>
		</tr>
		<tr>
			<td>Light Cycler Software</td>
			<td>Roche</td>
			<td>480 SW 1.5.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Modeling clay</td>
			<td></td>
			<td></td>
			<td>no specific provider</td>
		</tr>
		<tr>
			<td>King Fisher Flex Purification System</td>
			<td>ThermoFisher scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aedes albopictus</td>
			<td></td>
			<td></td>
			<td>eggs were collected in Freiburg, Germany</td>
		</tr>
		<tr>
			<td>Binocular</td>
			<td>MOTIC</td>
			<td>SMZ-168</td>
			<td></td>
		</tr>
		<tr>
			<td>Binocular light</td>
			<td>MOTIC</td>
			<td>MLC-150C</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2-Incubator</td>
			<td>Thermo scientific</td>
			<td>MIDI 40</td>
			<td></td>
		</tr>
	</tbody>
</table>

forced salivation as a method to analyze vector competence of mosquitoes

向量能力被定义为蚊子种类的潜力传播蚊子传播的病毒 (mobovirus) 到脊椎动物宿主。有效的病毒粒子通过感染蚊子的唾液在血液中传播。强迫流涎化验允许在单蚊子的基础上确定矢量电位, 避免使用动物实验。该方法适用于在短时间内对大量蚊虫进行一次实验分析。采用强迫流涎测定法分析了德国境内856只蚊子, 包括两种不同的淡色库蚊虱、淡色 torrentium和白纹伊蚊,实验感染与 Zika 病毒 (ZIKV) 和孵化在18°c 或27°c 为两个和三周。结果表明, 不同淡色类群对 ZIKV 的载体能力缺乏。相比之下,白纹伊蚊易受 ZIKV 的影响, 但只有27摄氏度, 传播速率类似于白纹伊蚊蚊实验室菌落平行测试。

白纹蚊被称为 ZIKV 的有效载体, 至少在热带气候条件下为12。因此, 我们使用Ae. 蚊作为一个积极的控制, 以建立检测和分析 IRs 以及在孵化温度为27°c 和湿度为80%。IR 和 TR 被定义为以前描述的财神等。13. 挑战Ae. 蚊显示了50% 和72% 的 IR 和42% 和31% 在14和21天后感染 (dpi), 分别 (图 1A和1B)。随后, 欧洲蚊子被测试 ZIKV 感染和传播使用热带和温带气候条件。
在27摄氏度的孵化温度下, 所有蚊子种类显示 ZIKV 感染在 14 dpi, 以及 21 dpi, 除了cx. 库蚊和cx. torrentium的个人, 这是积极的 14 dpi 仅。与不同的淡色类群相比, 所有的伊蚊物种都显示出较高的 irs (介于50% 和72% 之间) 以及较高的病毒 RNA 浓度 (106至 109 RNA 拷贝/蚊子标本), 这表明 irs 范围介于0% 和32% 和病毒装载 103到 104 RNA 拷贝或蚊子标本。所有的伊蚊物种都显示 ZIKV 阳性唾液 (TR 13–42%), 而淡色类群中的唾液样本没有 ZIKV (图 1A和1B)。有趣的是, 21 dpi 的传输速率在 ae 中是相似的. 蚊和ae从德国 (30%), 但极大地低在ae.来自意大利的伊蚊 (13%)。
在18摄氏度的适度温度下孵化有挑战的蚊子, 在白纹病毒的种类 (104–106 RNA 拷贝/蚊子) 以及在不同的淡色分类群 (102–104RNA 拷贝或蚊子)。然而, 在18摄氏度的孵化温度下, 没有观察到任何被调查的蚊子的 ZIKV 的传播。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57980/57980fig1.jpg"> 图 1: 实验性感染 ZIKV 的蚊子的传播速率.ZIKV 在不同蚊虫种类中的载体能力研究结果。六种不同的蚊子分类感染了 ZIKV, 在27摄氏度或18摄氏度处孵化 14 (A) 或 21 (B) 天。TR 被定义为蚊子的数量与 ZIKV 阳性唾液每数量 ZIKV 阳性蚊子身体。调查的蚊子总数为856人, 每个时间点/温度/蚊子分类的人数最少30。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57980/57980fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Forced Salivation As a Method to Analyze Vector Competence of Mosquitoes at JoVE.com

Forced Salivation As a Method to Analyze Vector Competence of Mosquitoes

强迫流涎法分析蚊虫的载体能力

为有效控制蚊媒传播病毒, 有关蚊子的矢量电位的知识尤其值得关注。本文将强迫流涎作为一种分析白纹伊蚊和三种不同淡色类群对 Zika 病毒传播的载体能力的方法。

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

forced-salivation-as-method-to-analyze-vector-competence

Nanyang Technological University

Research

JoVE Journal

Biology

9.5K 次观看.  Bernhard Nocht Institute for Tropical Medicine. 为有效控制蚊媒传播病毒, 有关蚊子的矢量电位的知识尤其值得关注。本文将强迫流涎作为一种分析白纹伊蚊和三种不同淡色类群对 Zika 病毒传播的载体能力的方法。

视频: Forced Salivation As a Method to Analyze Vector Competence of Mosquitoes

Injection of An. stephensi Embryos to Generate Malaria-resistant Mosquitoes

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This video protocol demonstrates a method used by the James laboratory to microinject DNA constructs into Anopheles stephensi embryos for the generation of transformed mosquitoes. Techniques for preparing microinjection needles, collecting and preparing embryos and performing the microinjection are illustrated.

Anopheles stephensi mosquitoes are vectors for malaria inhabiting India and throughout Asia. This video demonstrates the technique for performing microinjections of this species with transgenes that will confer resistance to the malaria to the mosquito. Much of the methodology demonstrated in this video is applicable to microinjection techniques of other mosquito species.

注射的。 stephensi胚胎生成抗药性疟疾的蚊子

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This ...

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 ...

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.

The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue rats.

走在大鼠作为定性分析方法使用Rotorod

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

斑马鱼的胚胎显微注射来分析基因的功能

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

脂质囊泡介导的亲和层析法，使用磁性细胞分选（LIMACS）:一种新的方法来分析蛋白质 - 脂质相互作用

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

A Multi-detection Assay for Malaria Transmitting Mosquitoes

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, several traits are typically characterized using molecular assays to aid in epidemiological studies. These traits include species identification, insecticide resistance, parasite infection status, and host preference. Since populations of the Anopheles gambiae complex are morphologically indistinguishable, a polymerase chain reaction (PCR) is traditionally used to identify species. Once the species is known, several downstream assays are routinely performed to elucidate further characteristics. For instance, mutations known as KDR in a para gene confer resistance against DDT and pyrethroid insecticides. Additionally, enzyme-linked immunosorbent assays (ELISAs) or Plasmodium parasite DNA detection PCR assays are used to detect parasites present in mosquito tissues. Lastly, a combination of PCR and restriction enzyme digests can be used to elucidate host preference (e.g., human vs. animal blood) by screening the mosquito bloodmeal for host-specific DNA. We have developed a multi-detection assay (MDA) that combines all of the aforementioned assays into a single multiplex reaction genotyping 33SNPs for 96 or 384 samples at a time. Because the MDA includes multiple markers for species, Plasmodium detection, and host blood identification, the likelihood of generating false positives or negatives is greatly reduced from previous assays that include only one marker per trait. This robust and simple assay can detect these key mosquito traits cost-effectively and in a fraction of the time of existing assays.

Malaria transmitting mosquitoes have a number of epidemiologically important characteristics that can only be detected using molecular techniques. Utilizing a MALDI-TOF based SNP genotyping platform, we developed an assay for simultaneously detecting multiple key traits (species, insecticide resistance, parasite infection and host choice) of malaria vectors.

一种多检测试验为疟疾蚊子发射

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, ...

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, ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

新型杀虫化学对蚊子毒性测试规程

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...

A Blood-Free Diet to Rear Anopheline Mosquitoes

Malaria research requires large-scale breeding and production conditions for mosquitoes (Anopheles spp.) in captivity. The sustainable and reliable production of mosquitoes is currently inhibited by the supply of fresh vertebrate blood. Alternatives to blood are required to promote efficient control strategies for malaria and other vector borne diseases that are transmitted by blood feeding insects. With this in mind, artificial liquid diets were formulated as substitutes for fresh vertebrate blood. Herein we report a blood-free artificial liquid diet that delivers feeding rates similar to blood and mimics the physiological effects of a fresh vertebrate blood meal. The diet induces ovarian and egg maturation of Anopheles mosquitoes and also produces good larval survival and development of functional adults. The formulated blood-free liquid diet is an important advance towards sustainable mosquito breeding in captivity and will reduce the maintenance costs of mosquito colonies and eliminate the need for fresh vertebrate blood.

A protocol is presented for formulation of a blood-free artificial diet to feed Anopheles mosquitoes in captivity. This diet has a similar performance to vertebrate blood and triggers oogenesis and egg maturation and produces viable adult progeny.

后尾性异草蚊子的无血饮食

Malaria research requires large-scale breeding and production conditions for mosquitoes (Anopheles spp.) in captivity. The sustainable and reliable ...

Glomerular Outgrowth as an Ex Vivo Assay to Analyze Pathways Involved in Parietal Epithelial Cell Activation

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of pathways involved in parietal epithelial cell activation could therefore be a tool to attenuate the progression of glomerular diseases. This article describes a method to culture and analyze parietal epithelial cell outgrowth of encapsulated glomeruli isolated from mouse kidney. After dissecting isolated mouse kidneys, the tissue is minced, and glomeruli are isolated by sieving. Encapsulated glomeruli are collected, and single glomeruli are cultured for 6 days to obtain glomerular outgrowth of parietal epithelial cells. During this period, parietal epithelial cell proliferation and migration can be analyzed by determining the cell number or the surface area of outgrowing cells. This assay can therefore be used as a tool to study the effects of an altered gene expression in transgenic- or knockout-mice or the effects of culture conditions on parietal epithelial cell growth characteristics and signaling. Using this method, important pathways involved in the process of parietal epithelial cell activation and consequently in glomerulosclerosis can be studied.

This article describes a method for culturing and analyzing glomerular parietal epithelial cell outgrowths of encapsulated glomeruli isolated from mouse kidney. This method can be used to study pathways involved in parietal epithelial cell proliferation and migration.

球状外生长作为前维沃分析分析涉及的路径的表皮上皮细胞激活

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of ...