Name: high-throughput functional screening using a homemade dual-glow luciferase assay
Uploaded: 2014-06-01T14:00:00
Description: Watch this Scientific Journal Video about High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay at JoVE.com

我们感谢在麻省总医院的DNA核心的约翰Darga制备的DNA筛查图书馆。 PerkinElmer公司的克里斯托弗Chigas为我们WALLAC 1420照度计的宝贵支持。史蒂芬Ciacco的和安捷伦的马丁Thomae规定的布拉沃机器人的支持。我们感谢罗恩·约翰逊和史蒂夫·提图斯在NIH慷慨地提供他们的双荧光素酶发光分析方案。

对于一个实验性的概述，参见图1。 1，准备报告质粒含有α-突触核蛋白启动子 α-突触核蛋白基因（ 图2）的调控元件跨度约10 kb的，从上游NACP（淀粉样肽的非A的β成分）二核苷酸重复序列1,2到2内含子3，我们在包括这个区域的一部分本报记者构造。我们发现，含有内含子2的成长需要特别程序质粒，概述如下。荧光?...

<ol>
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α-突触核蛋白有牵连的帕金森氏病（PD）作为路易体4，考虑特殊病征为疾病细胞内的夹杂物的成分。众多的全基因组关联研究已发现，单核苷酸多态性在α- ​​突触核蛋白与风险偶发性帕金森氏5,6,7增加。虽然比散发性PD不太常见，家族性帕金森病也可在α- ​​突触核蛋白8所造成的突变，以及复制和α-突触核蛋白基因9,10,11的三重，这种现象也出现在散发性PD ...

找出关键基因的调控元件，并在他们身上起作用的因素的能力是根本的为众多生物过程的探索。然而，识别，调节基因表达的稀有细胞类型的因素，如特定神经元群体，是具有挑战性的。在这里，我们提出了一个协议，用于识别α-突触核蛋白（SNCA）的新的转录调节，与帕金森氏症有关，表现在多巴胺能神经元在黑质的基因收杆中脑的致密区。我们使用的是高通量，双荧光素酶， 在体外记者屏幕解构α-突触核蛋白表达在293T细胞中实现这一点。 α-突触核蛋白的启动子是第一个克隆到含有萤火虫荧光素酶基因的报告质粒。含有组成型活性的启动子的控制下的海肾荧光素酶的市售质粒作为内部对照。成分股ë记者构建体共转染到293T细胞中微孔板与从DNA表达文库质粒，使得每孔转染了一个单一的文库质粒。 48小时后，对于每一个记者荧光素酶活性被依次使用双辉光测定法测量。在每个孔中的海肾荧光素酶的活性（F：R比率）板基于归一化后的α-突触核蛋白的响应于每个文库的基因的相对表达通过的萤火虫的比率推断。 该协议提供了筛选大量的基因（〜2,500每星期）就其使用最少的人力（1-2人），最低的成本（约300试剂费用为每微孔板检测），以式激活报告基因的能力的能力。神经细胞的基因的转录调节子（ 如 ，α-突触核蛋白），这是很难培养的神经元耐火遗传操作来研究，可以比在此直接功能测定并排侧。我们在包括我们协议的详细方法进行克隆和含有α-突触核蛋白的启动子的质粒的增长，因为我们发现，当用常规方法（见讨论）生长含有该区域的质粒是不稳定的。我们还包括了一种廉价的替代市售的双荧光素酶试剂，用于高通量测定法。这个协议可以很容易地适应目标的感兴趣的其它调控元件，或以任何需要高通量的瞬时转染过程。

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Material</td>
 </tr>
 <tr>
 <td>QIAQuick PCR Purification Kit</td>
 <td>Qiagen</td>
 <td>28106</td>
 <td></td>
 </tr>
 <tr>
 <td>PureLink Quick Gel Extraction Kit</td>
 <td>Invitrogen</td>
 <td>K2100-12</td>
 <td></td>
 </tr>
 <tr>
 <td>PureLink PCR Micro Kit</td>
 <td>Invitrogen</td>
 <td>K310250</td>
 <td></td>
 </tr>
 <tr>
 <td>PureLinkHiPure Plasmid Miniprep Kit</td>
 <td>Invitrogen</td>
 <td>K2100-03</td>
 <td></td>
 </tr>
 <tr>
 <td>PureLinkHiPure Plasmid Maxiprep Kit</td>
 <td>Invitrogen</td>
 <td>K2100-07</td>
 <td></td>
 </tr>
 <tr>
 <td>Bravo Robot</td>
 <td>Agilent</td>
 <td>-</td>
 <td></td>
 </tr>
 <tr>
 <td>Robot Pipet Tips with Filter</td>
 <td>Agilent</td>
 <td>19477-022</td>
 <td></td>
 </tr>
 <tr>
 <td>Robot Pipet Tips without Filter</td>
 <td>Agilent</td>
 <td>19477-002</td>
 <td></td>
 </tr>
 <tr>
 <td>Clear-bottomed 96-well Plates</td>
 <td>Corning</td>
 <td>3610</td>
 <td></td>
 </tr>
 <tr>
 <td>Reservoirs</td>
 <td>Axygen</td>
 <td>RES-SW96-HP-SI</td>
 <td></td>
 </tr>
 <tr>
 <td>Polystyrene V-bottomed 96-well Plate</td>
 <td>Greiner</td>
 <td>651101</td>
 <td></td>
 </tr>
 <tr>
 <td>Polypropylene V-bottomed 96-well Plate</td>
 <td>Greiner</td>
 <td>651201</td>
 <td></td>
 </tr>
 <tr>
 <td>Adhesive Plate Covers</td>
 <td>CryoStuff</td>
 <td>#FS100</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent</td>
 </tr>
 <tr>
 <td>Phusion DNA Polymerase</td>
 <td>New England Biolabs</td>
 <td>M0530L</td>
 <td></td>
 </tr>
 <tr>
 <td>BAC 2002-D6</td>
 <td>Invitrogen</td>
 <td>2002-D6</td>
 <td></td>
 </tr>
 <tr>
 <td>Calf Intestine Alkaline Phosphatase</td>
 <td>Roche</td>
 <td>10713023001</td>
 <td></td>
 </tr>
 <tr>
 <td>T4 DNA Ligase</td>
 <td>New England Biolabs</td>
 <td>M0202L</td>
 <td></td>
 </tr>
 <tr>
 <td>pGL4.10</td>
 <td>Promega</td>
 <td>E6651</td>
 <td></td>
 </tr>
 <tr>
 <td>pGL4.74</td>
 <td>Promega</td>
 <td>E6931</td>
 <td></td>
 </tr>
 <tr>
 <td>ElectroMAX Stbl4 Competent Cells</td>
 <td>Invitrogen</td>
 <td>11635-018</td>
 <td></td>
 </tr>
 <tr>
 <td>Subcloning Efficiency DH5&alpha; Competent Cells</td>
 <td>Invitrogen</td>
 <td>18265-017</td>
 <td></td>
 </tr>
 <tr>
 <td>DMEM without Phenol Red</td>
 <td>Invitrogen</td>
 <td>31053-028</td>
 <td></td>
 </tr>
 <tr>
 <td>Optifect</td>
 <td>Invitrogen</td>
 <td>12579017</td>
 <td></td>
 </tr>
 <tr>
 <td>Ciprofloxacin</td>
 <td>CellGro</td>
 <td>61-277-RF</td>
 <td></td>
 </tr>
 <tr>
 <td>Tris-Hydrochloride Powder</td>
 <td>Sigma</td>
 <td>93287</td>
 <td></td>
 </tr>
 <tr>
 <td>Tris-Base Powder </td>
 <td>Sigma</td>
 <td>93286</td>
 <td></td>
 </tr>
 <tr>
 <td>Triton X-100 Pure Liquid</td>
 <td>Fisher</td>
 <td>BP151-100</td>
 <td></td>
 </tr>
 <tr>
 <td>DTT</td>
 <td>Invitrogen</td>
 <td>15508-013</td>
 <td></td>
 </tr>
 <tr>
 <td>Coenzyme A</td>
 <td>Nanolight</td>
 <td>309</td>
 <td></td>
 </tr>
 <tr>
 <td>ATP</td>
 <td>Sigma</td>
 <td>A6419</td>
 <td></td>
 </tr>
 <tr>
 <td>Luciferin</td>
 <td>Invitrogen</td>
 <td>L2912</td>
 <td></td>
 </tr>
 <tr>
 <td>h-CTZ</td>
 <td>Nanolight</td>
 <td>301</td>
 <td></td>
 </tr>
 <tr>
 <td>PTC124</td>
 <td>SelleckBiochemicals</td>
 <td>S6003</td>
 <td></td>
 </tr>
 </tbody>
</table>

high-throughput functional screening using a homemade dual-glow luciferase assay

我们提出了一种快速，廉价的高通量筛选的协议，以确定α-突触核蛋白与帕金森病相关的基因的转录调节。 293T细胞瞬时转染从一个排列的ORF表达文库质粒连同荧光素酶报告质粒，在1个基因的每孔微孔板格式。 48小时，以确定每个基因库后的α-突触核蛋白的转录，归表达从内部控制结构（一hCMV启动子指导海肾萤光素酶）的影响后，萤火虫荧光素酶活性测定。该协议是由封闭在一个生物安全柜台式机器人，它在96孔板进行无菌液体处理提供便利。我们的自动转染协议很容易适应高通量慢病毒库生产或需要三转染的大量独特的文库质粒的共轭等功能筛查方案nction用一套共同辅助质粒。我们还提出一种廉价的和验证的替代市售的双荧光素酶试剂，它采用PTC124，EDTA和焦磷酸盐来抑制萤火虫荧光素酶活性之前， 海肾萤光素酶的测定。使用这些方法，我们筛选7,670人的基因并确定了68监管α-突触核蛋白。这个协议是很容易修改的目标感兴趣的其他基因。

典型的荧光素酶值，F：R比和为一个单一的半板的感应值被显示在图4中注井H2，32倍诱导的命中。基因会引起过度的毒性（例如，井E3），或井转染较差，会产生低的值既萤火虫和海肾萤光素酶，但平均感应值。基因会引起非特异性诱导荧光素酶活性，也许是通过与pGL4骨干相互作用，将导致萤火虫和海肾萤光素酶一样，产生的平均感应值。大的差异在重复之间的感?...

Watch this Scientific Journal Video about High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay at JoVE.com

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

我们目前确定采用高通量机器人转染和一个自制的双发光荧光素酶检测转录调控因子的快速和廉价的检查方法。该协议迅速产生直接并排端功能数据数以千计的基因，很容易修改针对任何感兴趣的基因。

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Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

手册小分子屏幕接近高通量使用斑马鱼胚胎

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

检测miRNA靶在高通量的使用3&#39;LIFE分析

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

qPCRTag分析 - 高通量，实时定量PCR检测方法的基因分型Sc2.0

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

激酶底物对鉴定使用高通量筛选


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...