Name: drug-induced sensitization of adenylyl cyclase: assay streamlining and miniaturization for small molecule and sirna screening applications
Uploaded: 2014-01-27T14:00:00
Description: Watch this Scientific Journal Video about Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications at JoVE.com

作者要感谢理查德·辛克先生和Todd Wiernicki方法学的培训和实验指导。我们还要感谢约翰·保罗·斯彭斯仔细阅读和编辑建议。这项工作是由卫生MH 060397，脑与行为研究基金会和美国礼来公司的研究所和公司通过礼来公司研究奖励计划（LRAP）的支持。

1。扩展含量就绪细胞和冷冻保存<ol><li>培养的CHO-K1-DRD 2L（CHO-D 2L）在15 cm 2的细胞培养皿中Ham氏F12培养基补充有1.0μML-谷氨酰胺，800微克/微升G418，300微克/微升潮霉素，100 U /微升细胞青霉素，100微克/微升链霉素和10％胎牛血清（FBS）。 </li><li>孵育的细胞在37℃下在湿润的培养箱中5％的CO 2，直至细胞为90-95％汇合。用10微升磷酸盐缓冲盐水（PBS）洗涤细胞，并通过加入3微?...

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I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+cAMP+BRET+sensor+to+characterize+a+novel+regulation+of+cAMP+by+the+sphingosine+1-phosphate/G13+pathway.">Use of a cAMP BRET sensor to characterize a novel regulation of cAMP by the sphingosine 1-phosphate/G13 pathway.</a> J. Biol. Chem. 282, 10576-10584 (2007).</li></ol>

在努力促进外源过敏的研究中，我们广泛地修改了我们以前的方法实现一个精简的“组合和阅读”格式，易于进行高通量筛选和行动检查机制。主要的修改我们的协议可以概括如下：1）使用冷冻保存的细胞作为“测定就绪”试剂;测定到384 - 孔格式的2）的小型化，并在HTRF测定3）的利用cAMP的。 采用冷冻保存我们的基于细胞的测定，大大提高了效率和可重复性为我们日常的实?...

一种自适应的腺苷酸环化酶（AC）信号的响应被称为异源或超敏的诺贝尔奖获得者，马歇尔·尼伦伯格博士的实验室首次发现。尼伦伯格博士提出，所观察到的交流增加下列反应慢性δ阿片受体的激活是一个从事鸦片耐受性和依赖性1的机制。除了 ​​慢性δ阿片受体激活，交流信号的这种神经适应性反应也发生以下几种其他Gαi/ O偶联受体2的持续激活。值得注意的是，许多这些受体与疼痛，神经精神及神经系统疾病有关，包括μ/κ阿片，D 2/4多巴胺，5HT 1A和M 2/4毒蕈碱受体2。除了尼伦伯格博士的研究结果，大量的证据存在连接交流信号的宣传，以慢性阿片受体激活都<e米&gt;在体外和体内 3-7。交流的致敏也与各种涉及D 2类多巴胺受体包括精神分裂症和帕金森氏病的疾病有关（综述见参考文献2）。尽管致敏的潜在重要性，精确的机制（S）与持久Gαi/邻 - 偶联受体的激活，导致增加的AC响应在很大程度上仍然未知相关联。 这些研究为研究对腺苷酸环化酶的致敏机制作为一项重要的神经生物学指标的理由。同样，AC的信令8的生理相关性和重要性，个人交流亚型在此适应性反应保持还应认识2,9,10。在我们的研究的情况下，使用交流的重组同种型异源致敏相关联的一般功能平行那些特征德cribed学习交流的内源亚型。具体来说，以前的研究已经发现，Gαi/ O蛋白质和随后发布/重排βγ亚基的激活是所有交流亚型受体引起过敏的重要要求。此外，一些研究表明，由蛋白激酶和Gβγ亚单位的信令中涉及致敏2,11-13。个别的AC也显示独特而鲜明的致敏模式12。举例来说，D 2受体的持续暴露于激动剂与AC1和AC8的致敏关联到的Ca 2 + /钙调蛋白刺激14,15，而与之密切相关的AC3不致敏2。 AC2，AC4和AC7有密切的关系，但是，只有PKC刺激AC2活动是D 2受体的长期接触后强劲致敏激动剂7,14,16,17。此外，AC5和AC6显示海特的显着程度ologous宣传，以天然气和毛喉素刺激的cAMP积累下激活D 2受体14,18-20，但出现在他们的Gβγ亚单位交流的要求，以不同的相互作用21。虽然交流致敏的大多数研究中使用的模型的细胞系（ 例如 HEK293细胞表达个人交流异构体），看来这些结果转化为原生神经元细胞模型4,22。最近，在HEK293细胞中表达的AC亚型鉴定AC亚型选择性的小分子抑制剂的效果，也可以转换为在体内的行为研究23。 的可识别的分子机制的异源致敏的缺乏可能反映了自适应响应的复杂性以及各个AC亚型12的独特的调节特性。揭开这种复杂性是由使用繁琐的方法吨进一步复杂化帽子已经从用人公正的途径有限的学术研究。例如，我们以前的机理研究涉及使用连续培养的细胞模型使用24 -和48 -孔组织培养格式15。培养的细胞通常培养48小时，然后进行激动剂药物治疗（2-18小时），随后通过一系列细胞洗涤和温育（ 图1）。 AC-同工型特异性的cAMP积聚的协议当时采用随后的cAMP积聚的测量使用一个费 ​​时费力的[3 H] cAMP结合的方法15,24。从开始的持续时间来完成每一测定通常总共四到五天需要从细胞接种到数据分析（ 图1）。新技术和自动化的应用导致了显着的增强功能在工业和HTS中心设置致敏研究。例如，一组具有国家中心的化学工作CAL基因组报道进行了两天的HTS检测程序确定的μ阿片受体引起过敏的小分子抑制剂在1,536孔板格式25。 本文章介绍了我们的努力，开发利用技术，可在大多数学术研究机构异源致敏研究高温超导实验。这种策略通过配合使用冻存细胞的细胞模型异源表达的D 2多巴胺受体与内源性或个人重组腺苷酸环化酶亚型（CHO-D 2L或HEK-AC6 / D 2 L）的组合来完成。为了提高我们的产量，我们重新设计了48和致敏试验（ 约 &gt; 20级以上4-5天）到384孔的格式，基本上是“混读”五步，一天检测。新的格式使用市售的同质时间分辨荧光（HTRF）测定来衡量的cAMP ACCumulation在完整细胞与多模式读板器。化验是稳健经得起small分子筛选，并可有效应用于筛选异源致敏抑制剂。此外，我们提供数据，允许使用该测定的siRNA的反向转染靶向或全基因组的siRNA文库筛选，只有轻微的修改，一般的方法。

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			<td height="28" style="height:28px;width:205px;">CHO-K1-DRD2L cells</td>
			<td style="width:144px;">DiscoveRx</td>
			<td style="width:148px;">930579C2</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Ham&#8217;s F12 media</td>
			<td style="width:144px;">VWR</td>
			<td style="width:148px;">SH30026fs</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Fetal Bovine Serum</td>
			<td style="width:144px;">VWR</td>
			<td style="width:148px;">SH3007003</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">G418</td>
			<td style="width:144px;">Sigma</td>
			<td style="width:148px;">A1720</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Hygromycin</td>
			<td style="width:144px;">Fisher</td>
			<td style="width:148px;">50-230-5556</td>
			<td></td>
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		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Penicillin/ streptomycin</td>
			<td style="width:144px;">Life Technologies</td>
			<td style="width:148px;">15070063</td>
			<td></td>
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		<tr height="28">
			<td height="28" style="height:28px;width:205px;">15 cm dishes</td>
			<td style="width:144px;">BD Falcon</td>
			<td style="width:148px;">353025</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">DMSO</td>
			<td style="width:144px;">Sigma</td>
			<td style="width:148px;">D2650</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Cell dissociation buffer</td>
			<td style="width:144px;">Life Technologies</td>
			<td style="width:148px;">13150016</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Phosphate Buffered Saline</td>
			<td style="width:144px;">Life Technologies</td>
			<td style="width:148px;">10010-049</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Cryovials</td>
			<td style="width:144px;">Fisher</td>
			<td style="width:148px;">976171</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Opti-MEM</td>
			<td style="width:144px;">Life Technologies</td>
			<td style="width:148px;">31985070</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">TC treated 384 well plates</td>
			<td style="width:144px;">Perkin-Elmer</td>
			<td style="width:148px;">6007688</td>
			<td></td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:205px;">HTRF cAMP Dynamic 2 kit</td>
			<td style="width:144px;">Cisbio Bioassays</td>
			<td style="width:148px;">62AM4PEB</td>
			<td style="width:199px;">http://www.htrf.com/camp-cell-based-assay</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Synergy 4</td>
			<td style="width:144px;">Biotek</td>
			<td style="width:148px;">H4MLFPTAD</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Prism 6</td>
			<td style="width:144px;">GraphPad Software</td>
			<td style="width:148px;">NA</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">3-Isobutyl-1-methylxanthine</td>
			<td style="width:144px;">Sigma</td>
			<td style="width:148px;">I5879</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">(&#177;)Quinpirole</td>
			<td style="width:144px;">Sigma</td>
			<td style="width:148px;">Q111</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Spiperone</td>
			<td style="width:144px;">Sigma</td>
			<td style="width:148px;">S7395</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Clozapine</td>
			<td style="width:144px;">Sigma</td>
			<td style="width:148px;">C6305</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Haloperidol</td>
			<td style="width:144px;">Sigma</td>
			<td style="width:148px;">H1512</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">S-(-)Sulpiride</td>
			<td style="width:144px;">Sigma</td>
			<td style="width:148px;">S112</td>
			<td></td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;width:205px;">Lipofectamine2000 transfection reagent</td>
			<td style="width:144px;">Invitrogen</td>
			<td style="width:148px;">11668019</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Trypan blue</td>
			<td style="width:144px;">Life Technologies</td>
			<td style="width:148px;">T10282</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Water, DNase, RNase-free</td>
			<td style="width:144px;">MP Biomedicals</td>
			<td style="width:148px;">821739</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Gas siRNA</td>
			<td style="width:144px;">Dharmacon</td>
			<td style="width:148px;">custom siRNA</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Non-targeting siRNA control</td>
			<td style="width:144px;">Dharmacon</td>
			<td style="width:148px;">D-001206-14-20</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">siGlo Red</td>
			<td style="width:144px;">Dharmacon</td>
			<td style="width:148px;">D-001630-02</td>
			<td></td>
		</tr>
	</tbody>
</table>

drug-induced sensitization of adenylyl cyclase: assay streamlining and miniaturization for small molecule and sirna screening applications

腺苷酸环化酶（AC）信号的致敏已经牵涉于多种神经精神和神经紊乱包括物质滥用和帕金森氏病。 Gαi/ O连接受体的急性激活抑制AC活性，而这些受体产生交流的异源致敏持续激活与细胞内cAMP水平的提高。以前的研究已经表明，这种增强的AC响应的观察在体外和体内以下几种类型Gαi/ O-连接的受体，包括D 2多巴胺和μ阿片受体的慢性活化。虽然交流的异源致敏首先报道四十年前，背后这一现象的机制（S）仍是未知。机械数据的缺乏可能反映的涉及本适应性反应的复杂性，这表明无偏差的方法可能有助于确定ING参与交流的异源致敏的分子途径。以前的研究已经牵连激酶和Gbγ信号的重叠，调节交流的异源致敏成分。标识与交流的过敏相关的独特的和额外的重叠的目标，一个可扩展的cAMP致敏试验开发和验证，需要更大的吞吐量。以前的方法来研究致敏一般都是笨重涉及连续细胞培养物保养以及一个复杂的方法用于测定cAMP的积累，涉及到多个洗涤步骤。因此，可在384孔的格式可用于高通量筛选（HTS）一个强大的基于细胞的测定的发展将有利于未来的研究。使用两个D 2多巴胺受体的细胞模型（ 即 ，CHO-D 2L和HEK-AC6 / D 2L），我们已经转换了48孔敏法（&gt; 20级4-5天），以五-STEP，在384孔板格式的单天测定。这种新格式是适合于小分子筛选，我们证明，该试验设计也可以容易地使用用于siRNA的反向转染在预期目标的siRNA文库筛选。

第一部分开发一个384以及异源致敏试验确定使用市售的细胞模型小分子抑制剂。 为了研究异源致敏的细胞模型，我们做了一些改进，使我们能够简化检测到一个“混合和阅读”格式。一些重要的修改包括以下各项，并且更详细地讨论所述。第一个关键步骤是从连续培养的细胞改变我们的细胞培养冻存细胞26。我们现在经常细胞培养批次可1-20×10 6?...

Watch this Scientific Journal Video about Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications at JoVE.com

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

持续激活的抑制性G蛋白偶联受体导致腺苷酸环化酶信令的致敏作用。以确定必需的分子途径，无偏差的方法是必要的，然而，这种策略需要一个可扩展的基于细胞的cAMP敏感测定法的发展。本文中，我们描述了一种致敏试验用于小分子和siRNA的筛选。

腺苷酸环化酶的药物引起的过敏性：含量简化和小型化的小分子和siRNA筛选应用

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

Research

JoVE Journal

Bioengineering

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

宽带超声医疗成像应用，光学检测仪

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic ...

科研

生物工程

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

在细胞和组织力学生物学应用一种新型的拉伸平台

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

多孔硅微粒进行交付的siRNA治疗的

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

固体脂质纳米粒（前哨淋巴结）细胞内定位的应用

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

一种快速定量荧光方法蛋白靶向小分子药物筛选

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

加工机械可调和生物活性金属支架的生物医学应用

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Preparation of Silicon Nanowire Field-effect Transistor for Chemical and Biosensing Applications

硅纳米线场效应晶体管的研制化学生物传感与应用

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

用于细胞培养应用光介导的形成和构图的水凝胶

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Extraction of Plant-based Capsules for Microencapsulation Applications

基于植物的提取胶囊微胶囊化应用

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin ...

Applications for Open Source Microplate-Compatible Illumination Panels

开源微孔板兼容照明面板的应用

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well ...