JoVE Journal > Cancer Research
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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암 연구학

使用阵列库的中等通量筛选和基于双 Luciferase 的分录报告器识别转录因子稳压器

Published: March 27, 2020
doi:
10.3791/60582
,
1Department of Molecular & Cellular Physiology,Albany Medical College

Summary

为了识别新的转录因子调节器，我们开发了一种使用基于双透明酶的转录报告器测定筛选排列的慢病毒或逆转录病毒RNAi库的方法。这种方法提供了一种快速且相对便宜的方法，在单个实验中筛选数百名候选人。

Abstract

转录因子可以改变许多目标基因的表达，这些基因影响各种下游过程，使它们成为抗癌治疗的良好目标。然而，直接瞄准转录因子通常是困难的，并可能导致不良的副作用，如果转录因子是必要的一个或多个成人组织。识别癌细胞中异常激活转录因子的上游调节器提供了一个更可行的替代方案，特别是如果这些蛋白质容易被药物。在这里，我们描述了一种协议，可用于组合阵列的中等尺度慢病毒库和基于双透明酶的转录报告器测定，以确定癌细胞转录因子的新调节器。我们的方法提供了一种快速、简单且廉价的方法，在单个实验中测试数百个基因。为了证明这种方法的使用，我们执行了一个阵列的慢病毒RNAi库的屏幕，该库包含几个调节器的Yes相关蛋白（YAP）和带PDZ结合图案（TAZ）的转录共活化器，两个转录共激活器，是Hippo通路的下游效应器。但是，这种方法可以修改为几乎所有转录因子或共因子的调节器进行筛选，也可用于筛选 CRISPR/CAS9、cDNA 或 ORF 库。

Introduction

此测定的目的是使用病毒库，以相对快速和廉价的方式识别转录因子的调节器。异常转录活性与癌症和转移11、2、3、4、5、62,3,4,5,6相关，因此靶向癌细胞中的转录因子是一种很有前途的治疗方法。然而，转录因子往往难以瞄准药理学7和许多需要正常细胞功能在成人组织8，9，10。9,108靶向癌症相关途径，异常激活转录因子驱动疾病是一种更可行的方法，有可能有不太严重的副作用。阵列慢病毒和逆转录病毒RNAi、CRISPR/CAS9、cDNA或ORF库的商业可用性使研究人员能够在单个实验中测试许多基因的重要性。但是，需要为更改的转录活动进行可靠的读出。

在这里，我们描述了使用基于双透明酶的转录报告器测定和排列的慢病毒库来识别调节癌细胞转录因子的蛋白质。在此测定中，针对癌症相关基因的shRNA通过慢病毒转导输送到哺乳动物癌细胞，并且选择细胞使用紫杉霉素进行稳定整合。细胞的下一个转染与一个记者构造，表达萤火虫透明酶驱动特定到被调查的转录因子和对照结构，表达雷尼拉透明化从一个产生活性的促进剂，对被调查的转录因子不响应。我们演示了这种方法，为YAP和TAZ的监管机构提供了概念验证屏幕，这是Hippo通路8、10、1110,的关键下游8效应器。11YAP和TAZ的异常活动促进转移级联11的几个步骤，并观察到在许多癌症11，12，13。11,12,13然而，YAP和TAZ如何变得异常激活在一些癌细胞还没有完全了解。YAP 和 TAZ 不绑定 DNA，而是通过其他转录因子招募给促进者。TEA 域 （TEAD） 转录因子系列的成员是 YAP 和 TAZ 的主要绑定合作伙伴，对于大多数 YAP 和 TAZ 相关函数至关重要。本报记者从YAP/TAZ-TEAD反应促进器中构建了萤火虫荧光酶，以往的研究表明，它忠实地检测了YAP-TEAD和TAZ-TEAD转录活动22、14、1514,15的变化。

我们的方法快速、中等吞吐量，不需要筛选设施、自动化机器人或池库的深层测序。成本相对较低，有许多商业上可用的图书馆可供选择。在大多数实验室中，所需的设备和试剂也相对标准。如果存在或生成基于荧光酶的分录器，它可用于筛选几乎任何转录因子的监管机构。我们使用这种方法在癌细胞中筛选shRNA，但任何能以合理效率转染的细胞系都可以用于任何类型的阵列库。

Protocol

注： 该协议的原理图摘要如图1所示。 1. 伦迪病毒病媒库制备 注：演示的屏幕使用在 96 孔板中作为甘油库存购买的阵列 shRNA 库，但库也可以根据候选列表手动组装。应考虑适当的控件并将其包含在任何库中。这包括非目标控制shRNA（shNTC），一种针对被调查的转录因子的控制shRNA，如果可能的话，一个针对萤火虫荧光酶的shRNA。 <ol…

Representative Results

我们的YAP/TAZ-TEAD记者构造 （pGL3-5xMCAT （SV）-492，,14，,15） 包含一个最小的SV-49启动器与5个重复的规范TEAD结合元素 （MCAT）15驱动萤火虫透明酶基因 （图1）。它与PRL-TK控制载体（Promega）一起被共转入细胞中，后者表示来自产生活性HSV TK促进剂的Renilla lucife…

Discussion

在这项研究中，我们演示了一种将基于双透明酶的转录器检测方法结合在一起，用于识别和测试新的转录因子调节器的中通量筛选方法。在任何屏幕之前，对每个单元行的分型和优化报告系统至关重要。应进行实验，以确认报告者对正在调查的转录因子的活动变化有反应，并且应对照对照载体测试活动变化程度。PRL-TK构造与报告器构造的共转导非常重要，因为它有助于控制与报告器一起转染的细?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们要感谢艾米莉·诺顿和米凯拉·库恰尔-斯图利格罗斯协助制备shRNA载体。这项工作得到了苏珊·科门职业催化剂赠款的部分支持，该赠款授予了J.M.L.（#CCR17477184）。

Materials

2.0 ml 96-well deep well polypropylene plate USA Scientific 1896-2000 For bacterial mini-prep
Trypsin – 2.50% Gibco 15090-046 Component of trypsin-EDTA
96 well flat bottom white assay plate Corning 3922 For dual-luciferase assay
Ampicillin – 100 mg/ml Sigma-Aldrich 45-10835242001-EA For bacterial mini-prep
Bacto-tryptone – powder Sigma-Aldrich 95039 Component of LB broth
Dual-luciferase reporter assay system, which include LAR II reagent (reagent A), Stop & Glo substrate (reagent B substrate) and Stop & Glo buffer (reagent B buffer) – Kit Promega E1960 For dual-luciferase assay
Dulbecco's phosphate buffered saline w/o calcium, magnesium and phenol red – 9.6 g/L Himedia TS1006 For PBS
EDTA – 0.5 M VWR 97061-406 Component of trypsin-EDTA
Ethanol – 100% Pharmco-AAPER 111000200 For bacterial mini-prep
Foetal Bovine Serum – 100% VWR 97068-085 Component of complete growth media
Hexadimethrine bromide (Polybrene) – 8 mg/ml Sigma-Aldrich 45-H9268 For virus infection
HyClone DMEM/High glucose – 4 mM L-Glutamine; 4500 mg/L glucose; sodium pyruvate GE Healthcare life sciences SH30243.01 Component of complete growth media
I3-P/i3 Multi-Mode Microplate/EA Molecular devices For dual-luciferase assay
L-Glutamine – 200 mM Gibco 25030-081 Component of complete growth media
Lipofectamine 3000 (Transfection Reagent 2) – 100% Life technologies L3000008 For transfections
Molecular Biology Water – 100% VWR 02-0201-0500 For dilution of shRNA vector for virus packaging
NaCl – powder BDH BDH9286 Component of LB broth
NanoDrop One Microvolume UV-Vis Spectrophotometer Thermo scientific For measuring vector DNA concentration
Opti-MEM (Transfection Buffer) – 100% Gibco 31985-062 For transfections
Penicillin Streptomycin – 10,000 Unit/ml (Penicillin); 10,000 µg/ml (Streptomycin) Gibco 15140-122 Component of complete growth media
PureLink Quick Plasmid Miniprep Kit – Kit Thermo Fisher Scientific K210010 For bacterial mini-prep
Puromycin – 2.5 mg/ml Sigma-Aldrich 45-P7255 For antibiotic selection after infection
TC20 automated cell counter Bio-Rad For cell counting
X-tremeGENE 9 DNA transfection reagent (Transfection Reagent 1) – 100% Roche 6365787001 For virus packaging
Yeast extract – powder VWR J850 Component of LB broth
P3000 (Transfection Reagent 3) – 100% Life technologies L3000008 For transfections
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Tags

Keywords: Transcription FactorMedium-throughput ScreeningArrayed LibrariesDual-luciferase ReporterLentiviral VectorRetroviral AssayBacterial Miniprep293FT CellsTransfectionPsPAX2VSVGCell Culture
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Xiao, Y., Lamar, J. M. Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter. J. Vis. Exp. (157), e60582, doi:10.3791/60582 (2020).
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