Name: rnai-mediated double gene knockdown and gustatory perception measurement in honey bees (apis mellifera)
Uploaded: 2013-07-25T08:55:00
Description: Watch this Scientific Journal Video about RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera at JoVE.com

笔者想，感谢Erin Fennern的协助实验。这项工作是由挪威研究理事会（180504，185306和191699），皮尤慈善信托基金，国家衰老研究所（NIA P01 AG22500）。

第1部分：RNAi介导的双基因敲除 1。双链RNA合成<ol><li>设计针对VG，USP和控制基因编码绿色荧光蛋白（GFP），这是不是在蜜蜂基因组的双链RNA引物设计引物，利用网上免费软件引物（ <a href="http://frodo.wi.mit.edu/" target="_blank">http://frodo.wi.mit.edu/</a> ） 。 </li><li>合成的双链RNA为VG，USP和GFP：利用RiboMax T7 RNA生产系统在体外转录自Promega。 </li><li>双链RNA纯...

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我们的研究是第一次努力，同时打掉两个基因在成虫。我们的研究结果表明，这两种策略（单次注射，为期两天的注射双链RNA注射）双基因抑制，同时基因沉默的VG和USP是有效的，可以测试后第6天的双链RNA注射到蜜蜂8,9。然而，基因敲除的功效不同的靶基因，蛋白质周转率和双链RNA的细胞或器官的吸收效率取决于转录水平不同的昆虫种类。此外，RNA干扰的效果是剂量依赖性?...

RNA干扰（RNAi）是基于RNA的转录后基因沉默，这发生在各种各样的真核生物。 RNAi过程被触发时由内源性或外源性的双链RNA（dsRNA）的前体。双链RNA激活核糖核酸酶蛋白Dicer酶结合并切割双链RNA小片段（20-25个基点）。然后，小片段的双链RNA导Argonaute蛋白的mRNA互补的识别和切割RNA诱导的沉默复合物（RISC），催化剂组分。在哺乳动物中，超过30 nt的双链RNA，激活抗病毒反应（IFN）干扰素反应，从而导致非特异性降解RNA转录2。然而，长dsRNA已被证明是有效的，具体的昆虫，因为有一个缺乏这IFN 3。 长双链RNA下调靶基因已被用于在不同的昆虫种类。蜜蜂是一个π介子能效比昆虫生物，其中许多重要功能基因的开发和行为已被揭露出来使用双链4,5。几个dsRNA的交付方法，已进行了蜜蜂的dsRNA喂养蜜蜂幼虫6有效下调靶基因的表达，而在蜜蜂胚胎和成年蜜蜂7,8双链RNA注射液是一种有效的基因敲除的方法。 展出的昆虫双链RNA的基因敲除的影响是短暂和局部的。有研究表明，腹腔注射双链RNA和胸双链RNA注射，有效地抑制靶基因的表达，腹部脂肪体细胞的昆虫9,10。双链RNA注射到腹腔和胸腔和脂肪体细胞能够采取双链RNA细胞血淋巴沐浴10。然而，基因在其他器官，如卵巢和大脑，不能有针对性要么腹腔或胸腔注射。以目标基因在蜜蜂的大脑，大脑的dsRNA注入也被执行，有效地影响局部脑区11靶基因的表达。在这里，我们只记录腹双链RNA注射，这是较为常用的成年蜜蜂。 RNA干扰已被主要用于针对一个单一的基因，一直是一个强大的工具来揭示基因功能。然而，任何基因从别人不是孤立的，它是在复杂的调控网络。了解生物过程的一个关键是解剖与对方，这需要同时操作多个基因，而不是一个单一的基因敲除基因如何相互作用。在哺乳动物细胞系同时抑制两种或三种基因，科学家们已经成功地通过输送系统12多的microRNA（miRNA）的发夹设计13。但在昆虫，多基因击倒仍未经验证。这里，我们PRESEN了吨不同注射的策略可以实现双基因敲除。我们的目标是两种基因：卵黄蛋白原（VG），它编码一个卵黄蛋白的前体，和ultraspiracle（ 美国药典 ），保幼激素（JH），编码一个假定的受体可能作为一种转录因子介导的反应在蜜蜂JH 14。 Vg和JH相互调节在反馈回路15，并参与了蜜蜂行为调节9。使用双基因敲除，我们扰乱Vg和JH途径，并发现它们是如何共同影响蜜蜂的行为和生理VG，USP和JH如何互动9。 味觉感知是一种行为预测蜜蜂社会行为16。行为的发展，巢蜜蜂高味觉感知行为成熟的快，通常在生命早期的牧草，喜欢收集花粉16,17。虽然监管米相关echanisms味觉感知目前仍不清楚，有研究表明，味觉的感知与内部能量代谢9，荷尔蒙分泌18,19和生物遗传胺通路20。 Vg和JH重要的荷尔蒙调节调节味觉感知7,21。在实验室中，味觉感知蜜蜂的变化进行评估测试长鼻延伸反应（PER）的不同的蔗糖溶液。每个蜂测试升序一系列浓度为0.1，0.3，1，3，10，30％的蔗糖的液滴的水通过触摸她的触角。注意，如果蜜蜂完全延伸她的长鼻液滴的水或蔗糖时，每个天线被触摸到的正响应。理想的反应溶液的数量的基础上，每一个人的味觉感知水平，可确定16。但是，应用程序的PER并不限于测量克感知ustatory。市盈率也是一种有效的方法来测试，如饱食与饥饿的代谢状态的蜜蜂。蜜蜂蔗糖有较大的反应一般是饿（王Amdam的，未发表的数据）。此外，市盈率的模式也可以被用来在联想学习和记忆的蜜蜂。在这种情况下，蜜蜂将接受培训，蔗糖水与气味是否存在关联。当蜜蜂学习的联想，只有存在的气味能唤起积极长鼻响应，没有奖励他们与蔗糖22,23。在这段视频中，我们将展示如何执行市盈率评估味觉感知，已与VG和USP双击倒在先前的研究9。

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>GE Healthcare PCR beads in 96 well plate</td>
			<td>GE Healthcare</td>
			<td>27-9557-01</td>
			<td></td>
		 </tr>
		 <tr>
			<td>QIAquick PCR purification kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td></td>
		 </tr>
		 <tr>
			<td>RiboMax T7 RNA production system</td>
			<td>Promega</td>
			<td>P1300</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trizol LS</td>
			<td>Invitrogen</td>
			<td>10296028</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Chloroform:Isoamyl alcohol 24:1</td>
			<td>Sigma-Aldrich</td>
			<td>C0549</td>
			<td></td>
		 </tr>
		 <tr>
			<td>isopropyl alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>I9516</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Hamilton micro syringe</td>
			<td>Hamilton</td>
			<td>80301</td>
			<td></td>
		 </tr>
		 <tr>
			<td>30G BD disposable needles</td>
			<td>BD Biosciences</td>
			<td>305106</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>84097</td>
			<td></td>
		 </tr>
		 <tr>
			<td>1 ml Syringe</td>
			<td>BD Biosciences</td>
			<td>30960</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Stereo dissection microscope</td>
			<td>Leica Microsystems</td>
			<td>S6D</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Insect pins</td>
			<td>Fine Science Tools</td>
			<td>26000-25</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Wax plate</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		</tbody>
 </table>

rnai-mediated double gene knockdown and gustatory perception measurement in honey bees (apis mellifera)

此视频演示新颖的RNA干扰（RNAi）技术下调两个基因同时使用双链RNA（dsRNA）的注射液的蜜蜂。同时还介绍了协议的长鼻扩展响应（PER）检测味觉感知测量。 RNAi介导的基因敲除技术是一种有效的下调靶基因的表达。这种技术通常用于单基因操作，但是它有局限性来检测基因之间的相互作用和共同作用。在这段视频的第一部分中，我们提出了两种策略，同时打掉两个基因的（称为双基因敲除）。我们表明这两种策略都能够有效地抑制这两个基因，卵黄蛋白原（VG）和ultraspiracle（USP），这是在调节反馈回路。这双基因敲除的方法可以用来分析基因之间的相互关系，可以很容易地应用在不同的昆虫种类。 该视频的第二部分是蜜蜂双基因敲除的治疗后长鼻扩展响应（PER）检测示范。市盈率法是一种标准的测试测量味觉感知的蜜蜂，这是一个重要的征兆，一个蜜蜂的行为成熟的速度有多快。大巢蜜蜂表示味觉感知行为发展的增加往往与花粉的觅食觅食专业化的发病年龄提前。此外，每次检测可用于识别代谢蜜蜂在饱食或饥饿状态。最后，结合配对不同的刺激气味，空调的蜜蜂也被广泛用于学习和记忆研究蜜蜂每次检测。

无论是单次注射，为期两天的注射策略明显减少VG（单因素方差分析，P &lt;0.001）（ 图2A）和USP（单因素方差分析，P &lt;0.001）（ 图2B）转录水平的蜜蜂6天之后的双链RNA注射。通过与双链RNA的混合物的单次注射和vg的第一和第二美国药典（vg的 / 美国药典 ）2天注射的美国药典转录抑制是显着低于两日的注射用美国药典 （ ?...

Watch this Scientific Journal Video about RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera at JoVE.com

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera

在这个协议中，我们描述了两种策略，同时抑制这两个基因在蜜蜂（双基因敲除）。然后，我们提出了如何用长鼻延伸反应（PER）的检测，以研究双基因敲除对蜜蜂的味觉感知的效果。

RNAi介导的双基因敲除和味觉感知测量蜜蜂（<em&gt;蜜蜂</em&gt;）

This video demonstrates novel techniques of RNA  interference (RNAi) which downregulate two genes simultaneously in honey bees  using double-stranded RNA ...

Research

JoVE Journal

Neuroscience

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Double Fluorescence in situ Hybridization in Fresh Brain Sections

双荧光原位杂交食脑切片

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

科研

神经科学

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees (Apis mellifera)

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees Apis mellifera

在蜜蜂的长鼻扩展响应的经典条件反射行为药理学（蜜蜂）

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability1,2. Because the ...

Creating Objects and Object Categories for Studying Perception and Perceptual Learning

创建对象和对象类别学习的认知与知觉学习

In order to quantitatively study object perception, be it perception by biological systems or by machines, one needs to create objects and object ...

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

慢性成人出生的嗅觉神经元的选择性病毒转导在体内 Optogenetic刺激

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

MPI CyberMotion Simulator: Implementation of a Novel Motion Simulator to Investigate Multisensory Path Integration in Three Dimensions

MPI Cyber​​Motion模拟器：一种新型的运动模拟器的执行情况调查三个维度的多感官的路径积分

Path integration is a process in which self-motion is integrated over time to obtain an estimate of one's current position relative to a starting point 1. ...

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees (Apis mellifera L.)

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees Apis mellifera L.

触觉空调和取样策略在蜜蜂触角的运动分析（<em&gt;蜜蜂</em&gt; L.）

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 2. The worker ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue

脑组织中的双管和同心微电极用于测量细胞外离子的信号

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular ...

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees (Bombus terrestris)

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees Bombus terrestris

一种新的行为分析调查个人的味觉，可自由移动的熊蜂（<em&gt;欧洲熊蜂</em&gt;）

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from ...

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

约束和自由飞行，蜜蜂的神经药理学操纵，<em&gt;蜜蜂</em

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize ...