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Medicine

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

Published: July 3rd, 2013

DOI:

10.3791/50369

1Department of Cell Biology, UT Southwestern Medical Center

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

A variety of luciferase-based reporters for monitoring a diverse array of cell biological processes are commercially available. The majority of these DNA constructs encode luciferase proteins that are induced to express by specific cell stimuli or perturbations. The most robust transcriptionally based reporters place the expression of a luciferase enzyme under the control of synthetic enhancer elements or gene promoter regions well-established for their reliability in reporting a physiologically relevant transcriptional event1,2. There are also trans-reporter luciferase systems that utilize GAL4-UAS to drive luciferase protein expression. Gal4 is a yeast tr....

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Entire protocol takes approximately 4 days.

1. Preparation of Cells for siRNA and Reporter Transfection

We will introduce here a protocol for interrogating siRNA libraries using a small set of siRNAs (384 different siRNA pools array in 96 well format with each pool consisting of 4x siRNAs targeting a single gene) from a genome-wide siRNA library for illustrative purposes. For this particular study, we will exploit HCT116 cells that exhibit deviant Wnt and Kras signali.......

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Despite advances in mapping the mutational landscape of various cancers using massive genome sequencing efforts12-14, we still do not have in place a systematic approach for translating these observations into intervention strategies. In colorectal cancer (CRC), mutations that are predicted to affect components of three cellular processes - Wnt/β-catenin, p53, and Kras signaling - are found in 99% of all tumors suggesting they are key drivers of cellular transformation in the gut13. Thus, cance.......

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There are several purveyors of luciferase-based reporter systems (such as Promega, Targeting Systems, New England BIolabs, and Thermo Fisher) that provide useful on-line resources for those entertaining a high-throughput screen for the first time. In addition, a number of excellent reviews regarding optimizing the use of high-throughput screens for gene discovery and how RNAi-based screening results can be integrated with other -omics-based datasets should be helpful15,16. Methods for the selection of "hits" f.......

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We acknowledge funding support from CPRIT (RP100119) and the Welch Foundation (I-1665).

....

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Name Company Catalog Number Comments
      REAGENTS
PathDetect Elk1 Trans-Reporting System Agilent Technologies Inc. 219005  
Pathway Profiling Luciferase System 4 pp53-TA-Luc Vector Clontech Lab Inc. 631914  
Effectene Transfection Reagent Qiagen Inc. 301427  
Dual-Luciferase Reporter Assay System Promega Corp. E1960  
Cypridina Luciferase Assay reagent Targeting Systems VLAR-1  
HCT116 cells ATCC CCL-247  
CytoTox-Fluo Cytotoxicity Assay Promega Corp. G9260  
      EQUIPMENT
MultiFlo Microplate Dispenser Labsystems Inc. Model 832  
Biomek FXP Laboratory Automation Workstation Beckman Coulter Inc. A31842  
PHERAstar FS-multi-mode HTS microplate reader BMG Labtech Inc. 0471-101A  
Bright-Line Reichert Hemacytometers Hausser Scientific Company 1490  

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