Name: high-throughput analysis of mammalian olfactory receptors: measurement of receptor activation via luciferase activity
Uploaded: 2014-06-02T14:15:00
Description: Watch this Scientific Journal Video about High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity at JoVE.com

这项工作是由R01 DC013339，R03 DC011373，和露丝属Kirschstein国家研究服务奖T32 DC000014支持。使用莫耐尔化学感受受体信号的核心，这是支持，部分由美国国立卫生研究院，NIDCD核心格兰特P30 DC011735资金进行的工作的一部分。作者感谢C. Sezille的帮助，收集数据。

Hana3A细胞1。文化<ol><li>通过补充的最小​​必需培养基（MEM），用10％（V / V）FBS的制备M10介质。 </li><li>文化维护<ol><li>维持细胞M10的媒体。注意：对于RTP1L，RTP2，REEP1和G表达载体αolf赋予Hana3A细胞嘌呤霉素抗性，但保持了细胞的这种抗生素没有显著影响测定结果。 </li><li>亚文化在1:8的10厘米菜每2-3天的比率。 </li><li>在37℃，5％CO 2。 </li></ol></li></ol> 2，用于转染?...

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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=Olfactory+Receptors.">Olfactory Receptors.</a> Olfactory Recept. Methods Protoc. 1003, 229-238 (2013).</li><li>Dey, S., Zhan, S., Matsunami, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+surface+expression+of+chemosensory+receptors+in+heterologous+cells.">Assaying surface expression of chemosensory receptors in heterologous cells.</a> Journal of visualized experiments JoVE. (48), (2011).</li></ol>

气味识别是通过嗅觉受体激活模式编码，但受体的激活模式，包括受体被激活到什么程度，被称为人类的嗅觉受体1-11少于6％。努力来表征嗅觉受体已经由劳动密集型方法或适用性不限于嗅觉受体家族17,23,24,33,34的一个子集。该Hana3A异源表达系统支持大多数测试嗅觉受体的鲁棒表达，并且可以在同一个的cAMP响应性荧光素酶报告系统，以监测嗅觉受体活化19,26,27一起使用。该...

哺乳动物嗅觉系统具有响应恶臭刺激广大，允许用于检测和数以千计的加臭剂的鉴别能力。嗅觉受体（OR值）是通过在嗅觉上皮12的嗅感觉神经元表达的分子传感器。哺乳动物的气味识别是通过激活不同的OR ​​通过的气味，而OR基因家族是广泛的，有大约1000小鼠和400人的受体12-16。在嗅觉神经元，并在异源细胞手术室以前的功能分析表明，不同的气味是由独特的认可，但重叠的OR ​​值10,17-20合奏。选配配体OR值是理解的嗅觉代码和建立嗅觉可行的模型必不可少的关键。由于表达OR值在异源系统，以及大量两者的气味和OR的困难，这一数据已经从F基本上不存在IELD;的确，人的口服补液盐少于6％已公布配体1-11。这个协议描述了使用荧光素酶测定法的表征气味/或相互作用。此法使手术室的高通量表征，任务是必不可少的理解气味/或相互作用以及发展气味编码的典范。 手术室的高通量研究面临的三大挑战。首先，表达的异源细胞的ORs被保留在ER中，并随后降解的蛋白酶体21,22，防止从OR值与在测定系统23-25 ​​气味物质相互作用。这个问题是由辅助蛋白，以促进广泛的ORs 19,26,27的稳定的细胞表面表达的发现处理。受体转运蛋白1和2（RTP1和2）促进或响应于刺激的气味19的细胞表面表达和活化。在此基础上，将HEK293T细胞修饰以稳定表达RTP1长（RTP1L）和RTP2，受体表达增强蛋白1和Gαolf，导致Hana3A细胞系19,27。此外，3型毒蕈碱型乙酰胆碱受体（M3-R）进行交互的ORs在细胞表面，并增强活化响应于加臭剂26。共转染的或用RTP1S和M3-R的进Hana3A细胞的结果在宽范围的ORs的在细胞表面27的健壮的，一致的，和功能性表达。第二，哺乳动物或剧目都相当大。在人类中，例如，或剧目是一个数量级比味觉受体库越来越多，2个数量级比视觉受体剧目更多了的。虽然克隆一个OR是一个相对简单的协议，需要显著的前期工作，以产生一个综合库。第三，虽然我们知道，在视觉，波长转换成颜色和在试音的频率转换成沥青，气味的组织是知之甚少，使得研究人员很难从气味物质的代表性样品进行插值。虽然有些已经取得了进展在这方面10,28，嗅觉景观的地图仍然是不完整的。筛选分子数万数百对口服补液盐是一项艰巨的任务;在这一领域的高通量筛选需要仔细界定的活动。其余的主要挑战是那些后勤和成本的，而不是固有的技术问题。虽然异源筛查还没有被广泛使用的学术团体，以确定配体，一间私人公司使用同样的技术来识别配体的100人的OR ​​值29。不幸的是，这些数据仍然是专有的。 这里所概述的高通量荧光素酶测定具有超过用于评估或激活的替代方法的几个优点。虽然respon本地嗅感觉神经元的SES使用电和钙成像进行了测量，这些技术有困难戏弄除了这或将导致由于对嗅觉神经元反应特性的重叠神经元的反应。虽然敲在GFP-标记的受体型30,31，通过腺病毒递送的特异性受体鼠嗅觉神经元32,33，或记录后进行RT-PCR 17,24,33可以连接到记录单受体类型，这些方法低通量和不适合于大型画面。异源筛查系统更具扩展性，以及两种主要形式被发现在文学：cAMP途径记者和三磷酸肌醇（IP3）途径记者。一旦刺激气味，OR值激活A Gαolf转导信号级联导致生产环磷酸腺苷（cAMP）的12。通过共转染交流电的控制下，萤火虫荧光素酶报告基因AMP应答元件（CRE），荧光素酶的生产可测气味响应的函数，允许进行定量或激活。或激活也可通过共表达G蛋白如Gα15/16或A Gα15-OLF嵌合体24,25,34相连的IP3通路。我们选择了这里提出的测定法基于以下三个因素：（1）共表达RTP1用的Rho-标记的嗅觉受体改善嗅觉受体在细胞表面19,27的表达; （2）使用的cAMP应答报告基因可用于测量或通过规范的第二信使通路的激活;和（3）的测定是非常适合于高通量筛选。 这样的高通量荧光素酶测定法是适用于各种研究有价值的嗅觉的字段。第一，大量的ORs可以确定一个SP受体激活图案进行筛选针对单一的加臭剂ecific气味。这种类型的研究发现OR7D4作为或负责响应类固醇气味雄8。相反，一种或可以以确定的受体响应曲线图10来筛选对加臭剂的一个面板。当候选嗅气味/或通过对这些屏幕识别，相互作用可以通过进行剂量反应实验检查或对气味的浓度增加的反应得到证实。剂量响应曲线也可以评估一个或遗传变异如何影响体外加臭剂的反应8,9,11,35，和这些研究可以扩展到种间OR变化，从而对受体的进化的跨物种和因果突变的进化的检查36,37，最后，此测定法可用于筛选气味拮抗剂能够拮抗或响应于对一个已知的加臭剂/受体对38,39的特定气味。总之，这种高通量的荧光素酶测定法是适用于一系列的研究，这将有助于表征或激活模式，并提供一个更好的理解气味编码的嗅觉系统。

<table border="0" cellpadding="0" cellspacing="0" style="width:685px;" width="685">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Name of Material/ Equipment</td>
			<td style="width:135px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:265px;">Hana3A cells</td>
			<td style="width:135px;">Avaiable from the Matsunami Laboratory upon request</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:265px;">RTP1S-pCI</td>
			<td style="width:135px;">Avaiable from the Matsunami Laboratory upon request</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:265px;">M3-R-pCI</td>
			<td style="width:135px;">Avaiable from the Matsunami Laboratory upon request</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">pCRE-luc</td>
			<td style="width:135px;">Agilent</td>
			<td style="width:119px;">219076</td>
			<td><a href="http://www.chem.agilent.com/store/ProductDetail.aspx?productID=219076%28Agilent%29">LUC</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">pSV40-RL</td>
			<td style="width:135px;">Promega</td>
			<td style="width:119px;">E2231</td>
			<td><a href="http://www.promega.com/products/reporter-assays-and-transfection/reporter-vectors-and-cell-lines/prl-renilla-luciferase-control-reporter-vectors/">RL</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Minimum Essential Media, Eagle</td>
			<td>Sigma Aldrich</td>
			<td>M4655</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/m4655?lang=en&amp;region=US">MEM</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">FBS</td>
			<td>Life Technologies</td>
			<td>16000-044</td>
			<td><a href="https://products.invitrogen.com/ivgn/product/16000044?ICID=search-16000044">FBS</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS (without Ca2+ and Mg2+)</td>
			<td>Cellgro</td>
			<td>21-040-CV</td>
			<td><a href="http://cellgro.com/products/balanced-salt-solutions/phosphate-buffered-saline/phosphate-buffered-saline-1x-2.html">PBS</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin (0.05% Trypsin EDTA)</td>
			<td>Life Technologies</td>
			<td>25300</td>
			<td><a href="https://products.invitrogen.com/ivgn/product/25300054?ICID=search-product">Trypsin</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">CD293</td>
			<td>Life Technologies</td>
			<td>11913-019</td>
			<td><a href="http://products.invitrogen.com/ivgn/product/11913019?ICID=search-11913019">CD293</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">96 well PDL white/clear plate</td>
			<td style="width:135px;">BD BioCoat</td>
			<td style="width:119px;">356693</td>
			<td><a href="http://www.thomassci.com/Supplies/Tissue-Culture-Cellware/_/BD-Biocoat-Poly-D-Lysine-Culture-Dishes?q=356693">plates</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:265px;">Lipid transfection reagent: Lipofectamine 2000</td>
			<td>Life Technologies</td>
			<td>11668-019</td>
			<td><a href="https://products.invitrogen.com/ivgn/product/11668019?ICID=search-11668019">Lipofectamine</a></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:265px;">Firefly luciferase substrate, firefly luciferase quencher/Renilla luciferase substrate: Dual-Glo Assay</td>
			<td style="width:135px;">Promega</td>
			<td style="width:119px;">E2980</td>
			<td><a href="http://www.promega.com/products/reporter-assays-and-transfection/reporter-assays/dual_glo-luciferase-assay-system/?__utma=1.1932750276.1376486173.1376486173.1376486173.1&amp;__utmb=1.1.10.1376486173&amp;__utmc=1&amp;__utmx=-&amp;__utmz=1.1376486173.1.1.utmcsr=google|utmc">dual glo</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Synergy S2&#160;</td>
			<td style="width:135px;">BioTek</td>
			<td style="width:119px;">SLAD</td>
			<td><a href="http://www.biotek.com/products/microplate_detection/synergy2_multimode_microplate_reader.html">BioTek S2</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:265px;">Microplate reader software: Gen5 Data Analysis Software</td>
			<td style="width:135px;">BioTek</td>
			<td style="width:119px;">Gen5</td>
			<td><a href="http://www.biotek.com/products/microplate_software/gen5_data_analysis_software.html">Gen5</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">BIOSTACK</td>
			<td style="width:135px;">BioTek</td>
			<td style="width:119px;">BIOSTACK2WR</td>
			<td><a href="http://www.biotek.com/products/microplate_software/biostack_microplate_stacker.html">BioStack</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Multiflo</td>
			<td style="width:135px;">BioTek</td>
			<td style="width:119px;">MFP</td>
			<td><a href="http://www.biotek.com/products/liquid_handling/multiflo_microplate_dispenser.html">MultiFlo</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">300ul GripTips</td>
			<td style="width:135px;">Integra</td>
			<td style="width:119px;">4433</td>
			<td><a href="http://www.integra-biosciences.com/sites/us/pipette_tips_griptips_4_e.html">GripTips</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">12.5ul GripTips</td>
			<td style="width:135px;">Integra</td>
			<td style="width:119px;">4414</td>
			<td><a href="http://www.integra-biosciences.com/sites/us/pipette_tips_griptips_4_e.html">GripTips</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">300ul GripTips ViaFlo96</td>
			<td style="width:135px;">Integra</td>
			<td style="width:119px;">6433</td>
			<td><a href="http://www.integra-biosciences.com/sites/us/pipette_tips_griptips_XYZ_4_e.html">XYZ tips</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">12.5ul GripTips 384 XYZ</td>
			<td style="width:135px;">Integra</td>
			<td style="width:119px;">6403</td>
			<td><a href="http://www.integra-biosciences.com/sites/us/pipette_tips_griptips_XYZ_4_e.html">XYZ tips</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">384ViaFlo</td>
			<td style="width:135px;">Integra</td>
			<td>6030</td>
			<td><a href="http://www.integra-biosciences.com/sites/us/96_384_channel_pipette.html">384ViaFlo</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TE buffer</td>
			<td>Macherey Nagel</td>
			<td>740797.1</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td>Sigma Aldrich</td>
			<td>D2650-100ML</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/d2650?lang=en&amp;region=US">DMSO</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">forskolin</td>
			<td>Enzo Life Sciences</td>
			<td>BML-CN100-0010</td>
			<td><a href="http://www.thomassci.com/007635F0-AA41-4B24-B46A-05723D685997/_/FORSKOLIN-10MG?q=c002e24">FOR</a></td>
		</tr>
	</tbody>
</table>

high-throughput analysis of mammalian olfactory receptors: measurement of receptor activation via luciferase activity

创建的气味嗅觉受体激活的独特和重叠的图案，让一个家庭的约1,000名鼠和400人的受体识别数以千计的气味的。加臭剂的配体已经公布人类受体1-11少于6％。这种缺乏数据的部分原因是由于功能上的困难表达这些受体在异源系统。在这里，我们描述了用于表达大多数嗅觉受体家族中Hana3A细胞，然后用荧光素酶报告基因分析嗅觉受体活化的高通量评估的方法。此法可用于（1）屏幕对嗅觉受体面板的气味的面板; （2）通过剂量反应曲线确定气味/受体相互作用;和（3）中的受体的变体比较受体的激活水平。在我们的样本数据，328的嗅觉受体进行了筛选对26的气味。气味/受体对具有不同的反应得分均选择位泰德和剂量反应测试。这些数据表明，一个屏幕是丰富对加臭剂/受体对，将通过剂量反应实验， 即受体具有一种加臭剂善意响应的有效方法。因此，这种高通量荧光素酶测定法是表征嗅觉受体 - 一个基本朝向气味编码在哺乳动物嗅觉系统的一个模型的步骤的一种有效方法。

一个主屏幕测试328对手术室26气味在100μm的浓度。这种气味浓度已被证明有效激活OR值与已知配体10的相当大的比例。首先，标准化的荧光素酶活性计算方式为将萤火虫荧光素酶的阅读由海肾萤光素酶的阅读。接着，基线值的计算中减去归一化的荧光素酶的读数用于从归一化的荧光素酶的读数没有气味控制每个孔（ 图1）。剂量反应曲线上48加臭剂进行与/或对随机分布在?...

Watch this Scientific Journal Video about High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity at JoVE.com

High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity

嗅觉受体的激活模式进行编码气味的身份，但缺乏公布的数据识别气味的配体哺乳动物的嗅觉受体的阻碍气味编码进行了全面的发展模式。这个协议描述了一种方法，以促进嗅觉受体的配体和受体活化的定量高通量鉴定。

受体激活的测量通过荧光素酶活性：哺乳动物的嗅觉受体的高通量分析

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors ...

Research

JoVE Journal

Neuroscience

Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

切片的制备，组织器官型培养，并记录在视交叉上核时钟基因活性的荧光素酶

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the ...

科研

神经科学

In vivo Electroporation of Developing Mouse Retina

In vivo Electroporation of Developing Mouse Retina

在发展中国家小鼠视网膜体内电穿孔

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

从使用吸吸液管技术的嗅觉受体神经元的气味引起的反应记录

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

成人下丘脑神经发生局灶性放射性抑制功能审讯

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral ...

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 ...

FIM Imaging and FIMtrack: Two New Tools Allowing High-throughput and Cost Effective Locomotion Analysis

FIM成像和FIMtrack：两个新的工具允许高吞吐量和成本效益分析步态

The analysis of neuronal network function requires a reliable measurement of behavioral traits. Since the behavior of freely moving animals is variable to ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

鼠标嗅觉感官神经元的完整神经上皮穿孔膜片钳记录：神经元表达鉴定的气味受体功能分析

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors

受体放射自显影协议血管紧张素Ⅱ受体的本地化的可视化

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to ...

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay

实时 cAMP 检测气味受体激活的活细胞测量

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. ...