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

Summary

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.

Abstract

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.

Introduction

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 event^1,2. There are also trans-reporter luciferase systems that utilize GAL4-UAS to drive luciferase protein expression. Gal4 is a yeast tr....

Protocol

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 signaling, and p53 activity. The appropriate cell line in studies aimed at interrogating other cellular processes of interest will have to be identified and ....

Results

Despite advances in mapping the mutational landscape of various cancers using massive genome sequencing efforts^12-14, 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, cancer gen.......

Discussion

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 helpful^15,16. Methods for the selection of "hits" f.......

Materials

List of materials used in this article

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