Name: a multiplexed luciferase-based screening platform for interrogating cancer-associated signal transduction in cultured cells
Uploaded: 2013-07-03T13:00:00
Description: Watch this Scientific Journal Video about A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells at JoVE.com

我们承认资助CPRIT（RP100119）和韦尔奇基金会（I-1665）。

整个协议约需4天。 1。制备的siRNA和记者转染的细胞我们将在这里介绍使用一小部分的siRNA（384不同的siRNA池阵列每个池组成的4倍siRNA靶向单个基因在96孔板格式）从全基因组siRNA文库，仅供询问的siRNA库的协议。对于这个特定的研究中，我们将利用HCT116细胞表现出离经叛道的Wnt和KRAS信号，p53的活性。合适的细胞系的研究，旨在询问其他细胞的过程，还必须?...

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有几个传播者基于荧光素酶报告系统（如Promega公司，瞄准系统，New England Biolabs公司，赛默飞世尔）的上线提供了有用的资源，对于那些第一次招待的高通量筛选。此外，一些优秀的评论关于优化利用高通量筛选基因的发现，以及如何基于RNAi筛查结果可以集成其他组学为基础的数据集应该是有帮助的15,16。 “点击”高通量筛选方法的选择是另一个讨论的主题是超出了本报告的范围。关于?...

监测多样化的细胞的生物过程的各种荧光素酶为基础的记者，是市售的。这些DNA构建的大部分编码荧光素酶蛋白质的诱导表达特定细胞刺激或扰动。最强大的以转录为基础的记者将表达荧光素酶的酶合成的增强子元件或建立对他们的可靠性报告生理上相关的转录事件1,2的基因的启动子区的控制之下。也有记者反荧光素酶系统，​​利用GAL4-UAS驱动荧光素酶蛋白的表达。 Gal4的是一种酵母转录激活因子，在UAS增强子的Gal4的特异性结合以激活转录的基因序列放置3向下流。这些类型的荧光素酶系统通常支持了DNA质粒 - 一个编码的GAL4融合蛋白的调控Ry的感兴趣的蛋白部分，和第二编码的控制下的一个或更多的UAS序列下的荧光素酶的蛋白质。因此，细胞的荧光素酶的信号反映感兴趣的蛋白质的活性水平。基于荧光素酶的记者的另一个常见用途是用于监测目的蛋白的融合蛋白的稳定性，即荧光素酶4。不管记者的类型，在这里指出，作为一个可以不承担任何记者的特异性已审问一个买者自负。因此，尽职调查是必需的任何研究战略纳入新记者构造。 读出的荧光素酶的选择可以是一样重要的控制其表达的DNA元件的可靠性。鉴于萤火虫荧光素酶（佛罗里达州）是最常用的酶在市售的结构，出现新的荧光素酶报告系统，不需要ATP（为的情况下佛罗里达州基反应），或者采用更稳定的酶，发出更强的信号，承诺这个研究平台，以提高效率和可靠性。荧光素酶的记者关于选择化学研究中一个重要的注意事项是佛罗里达州的易感性化学抑制，可能会引起虚假的报告。根据我们的经验，其他酶如海肾或荧光素酶Gaussia（RL和GL），分别利用腔肠基板不易抑制小分子5。 复用荧光素酶读出的最常见的格式，主要是因为可获得的易于使用的工具包，顺序地从单一样品中酶活性的测量的能力，需要使用FL和RL。在大多数试剂盒，样品第一次接触到荧光素揭示佛罗里达州的活动，然后通过淬火试剂同时沉积腔肠素产生次生次生RL信号。与另外的其他的荧光素酶可分泌，如GL或，使用另一种基片，如海萤荧光素酶（CL），更大数量的可能性，用于产生高的内容数据，使用荧光素酶已经出现。可以在这里找到6全基因组RNAi筛选使用这种技术的一个例子。这些技术进步反过来刺激特定的机制来解渴，以限制串扰7每种类型的荧光素酶的发展。在我们的手中，我们注意到一个更大的频率的“边缘效应”（莫名的趋势与边缘的高滴度文化板相关的细胞的活性），如GL或CL分泌荧光素酶。另一方面，例如分泌的荧光素酶动力学分析是有用的，因为它们使信号采样不掺细胞活力。 这里介绍我们的研究中，我们将同时监测三个癌症有关的活动细胞过程：P53，KRAS，Wnt信号转导通路。在我们的研究中成立记者pp53-TA-吕克FL记者从Clontech公司（以下简称为P53-FL记者）8，一个的记者Elk1-GAL4/UAS-CL系统（以下简称ELK-1记者安捷伦）， 8X的TCF RL记者，集成了多种合成的增强子元件Wnt信号通路的转录调控因子被称为T细胞因子（或粤港科技合作资助计划）9-11确认。

<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>PathDetect Elk1 Trans-Reporting System</td>
			<td>Agilent Technologies Inc.</td>
			<td>219005</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pathway Profiling Luciferase System 4 pp53-TA-Luc Vector</td>
			<td>Clontech Lab Inc.</td>
			<td>631914</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Effectene Transfection Reagent</td>
			<td>Qiagen Inc.</td>
			<td>301427</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dual-Luciferase Reporter Assay System</td>
			<td>Promega Corp.</td>
			<td>E1960</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cypridina Luciferase Assay reagent</td>
			<td>Targeting Systems</td>
			<td>VLAR-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HCT116 cells</td>
			<td>ATCC</td>
			<td>CCL-247</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CytoTox-Fluo Cytotoxicity Assay</td>
			<td>Promega Corp.</td>
			<td>G9260</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>MultiFlo Microplate Dispenser</td>
			<td>Labsystems Inc.</td>
			<td>Model 832</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Biomek FXP Laboratory Automation Workstation</td>
			<td>Beckman Coulter Inc.</td>
			<td>A31842</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PHERAstar FS-multi-mode HTS microplate reader</td>
			<td>BMG Labtech Inc.</td>
			<td>0471-101A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bright-Line Reichert Hemacytometers</td>
			<td>Hausser Scientific Company</td>
			<td>1490</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

a multiplexed luciferase-based screening platform for interrogating cancer-associated signal transduction in cultured cells

使用RNA干扰（RNAi）的基因的功能基因组规模的审讯化学听话癌细胞漏洞的快速鉴定具有很大的前景。限制这一技术的潜力是无法迅速划定机理的基础表型的结果，从而告知的发展，分子靶向治疗策略。我们在这里概括的方法来解构细胞表型诱导RNAi介导的基因打靶使用多路记者关键癌症细胞相关的流程监控系统，使。高内涵筛选方法是通用的，可以很容易地适用于其他类型的大型分子库的筛选。

尽管绘制各种癌症的突变景观采用大规模的基因组测序工作12-14的进步，我们仍然不到位有一个系统的方法将这些意见纳入干预战略。结直肠癌（CRC），预测的突变，影响组件的三个细胞过程 - Wnt基因/β-catenin蛋白，P53，和KRAS信号 - 被发现在所有肿瘤的99％，这表明他们是在肠道细胞转化的关键驱动因素13。因此，癌症基因组数据集可能丰富支持共同点促癌过程的基因，并可能被?...

Watch this Scientific Journal Video about A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells at JoVE.com

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

实现系统级的细胞过程的理解，是现代细胞生物学的目标。我们在这里描述的各种细胞功能的复用荧光素酶记者的战略点审问基因的功能基因组规模的RNAi库。

多路复用讯问癌症相关的信号转导在培养的细胞中的荧光素酶的筛选平台

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Research

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Medicine

How to Measure Cortical Folding from MR Images: a Step-by-Step Tutorial to Compute Local Gyrification Index

如何从MR图像测量皮质折叠：一个循序渐进的教程，以计算本地Gyrification指数

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will ...

科研

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High Content Screening in Neurodegenerative Diseases

在神经退行性疾病的高内涵筛选

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be ...

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

的的膀胱测压小老鼠：A膀胱Chemosensation研究的使用

	The lower urinary tract (LUT) functions as a  dynamic reservoir that is able to store urine and to efficiently expel it at a  convenient time. While ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

对黑色素瘤修饰符一个斑马鱼原地肿瘤模型的筛选

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

Voluntary Breath-hold Technique for Reducing Heart Dose in Left Breast Radiotherapy

自愿屏气技术减少心脏剂量的左乳房放射治疗

Breath-holding techniques reduce the amount of radiation received by cardiac structures during tangential-field left breast radiotherapy. With these ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

颈动脉输液的紫杉烷类化合物在小鼠体内的药代动力学和药效学分析

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

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高含量<em&gt;体外</em&gt;胰岛β细胞复制发现平台

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling

筛选生物活性纳米颗粒在噬菌体免疫细胞中的Toll样受体信号传导抑制剂

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have ...

Routine Screening Method for Microparticles in Platelet Transfusions

血小板输血中微粒的常规筛选方法

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood ...