Published: July 3rd, 2013
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....
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.......
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.......
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.......
|PathDetect Elk1 Trans-Reporting System
|Agilent Technologies Inc.
|Pathway Profiling Luciferase System 4 pp53-TA-Luc Vector
|Clontech Lab Inc.
|Effectene Transfection Reagent
|Dual-Luciferase Reporter Assay System
|Cypridina Luciferase Assay reagent
|CytoTox-Fluo Cytotoxicity Assay
|MultiFlo Microplate Dispenser
|Biomek FXP Laboratory Automation Workstation
|Beckman Coulter Inc.
|PHERAstar FS-multi-mode HTS microplate reader
|BMG Labtech Inc.
|Bright-Line Reichert Hemacytometers
|Hausser Scientific Company
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