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

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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Genet. 19, 379-383 (1998).</li><li>Korinek, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+members+of+the+Tcf+family+implicated+in+Wnt/beta-catenin+signaling+during+embryogenesis+in+the+mouse.">Two members of the Tcf family implicated in Wnt/beta-catenin signaling during embryogenesis in the mouse.</a> Mol. Cell Biol. 18, 1248-1256 (1998).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+molecular+characterization+of+human+colon+and+rectal+cancer.">Comprehensive molecular characterization of human colon and rectal cancer.</a> Nature. 487, 330-337 (2012).</li><li>Koboldt, D. 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O.K. and L.L are authors on a pending patent relating to multiplexed luciferase technology.

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...

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 transcriptional activator and the UAS is an enhancer to which Gal4 specifically binds to activate the transcription of gene sequences placed down-stream of it3. These types of luciferase systems are typically supported by two DNA plasmids - one encodes the GAL4 protein fused to the regulatory portion of a protein of interest, and the second encodes a luciferase protein placed under the control of one or more UAS sequences. Thus, the cellular luciferase signal reflects the activity level of the protein of interest. Another common use of luciferase-based reporters is for monitoring protein stability whereby a protein of interest is fused to a luciferase enzyme4. Regardless of the type of reporter, a caveat emptor is noted here as one cannot assume the specificity of any reporter to have been well interrogated. Thus, due diligence is required in the incorporation of new reporter constructs in any research strategy.
The selection of the luciferase enzyme read-out can be as important as the reliability of the DNA elements that control its expression. Whereas firefly luciferase (FL) is the most commonly used enzyme in commercially available constructs, the advent of new luciferase reporter systems that do not require ATP (as in the case of FL-based reactions) or that incorporate more stable enzymes that emit stronger signals promise to improve the efficiency and reliability of this research platform. An important note regarding the selection of luciferase reporters in chemical studies is the susceptibility of FL to chemical inhibition that could give rise to false reports. In our experience, other enzymes such as Renilla or Gaussia luciferase (RL and GL, respectively) that utilize coelenterazine as a substrate are less readily inhibited by small molecules5.
The most common format for multiplexing luciferase read-outs entails the use of FL and RL largely given the availability of easy-to-use kits with the ability to sequentially measure enzymatic activities from a single sample. In most kits, samples are first exposed to luciferin to reveal levels of FL activity followed by a quenching reagent that is simultaneously deposited with coelenterazine to yield a secondary RL signal. With the addition of other luciferases that can be secreted such as GL or that use yet another substrate such as Cypridina luciferase (CL), a greater number of possibilities for generating high content data using luciferases have emerged. An example of a genome-wide RNAi screen using this technique can be found here6. These technological advances have in turn spurred the development of specific mechanisms to quench each type of luciferase enzyme in order to limit cross-talk7. In our hands, we note a greater frequency of "edge effects" (inexplicable trends in cellular activity associated with the edge of high titer culture plates), with secreted luciferases such as GL or CL. On the other hand, such secreted luciferases are useful for kinetic assays as they enable signal sampling without adulterating cell viability.
For our study presented here, we will simultaneously monitor the activity of three cancer-relevant cellular processes: the p53, Kras, and Wnt signal transduction pathways. The reporters incorporated in our study are the pp53-TA-Luc FL reporter from Clontech (henceforth referred to as the p53-FL reporter)8, an Elk1-GAL4/UAS-CL reporter system (henceforth Elk-1 reporter; Agilent), and the 8X TCF RL reporter that incorporates multiple synthetic enhancer elements recognized by the Wnt pathway transcriptional regulators known as T-cell factors (or TCFs)9-11.

<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>PathDetect Elk1 Trans-Reporting System</td>
			<td>Agilent Technologies Inc.</td>
			<td>219005</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pathway Profiling Luciferase System 4 pp53-TA-Luc Vector</td>
			<td>Clontech Lab Inc.</td>
			<td>631914</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Effectene Transfection Reagent</td>
			<td>Qiagen Inc.</td>
			<td>301427</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dual-Luciferase Reporter Assay System</td>
			<td>Promega Corp.</td>
			<td>E1960</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cypridina Luciferase Assay reagent</td>
			<td>Targeting Systems</td>
			<td>VLAR-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HCT116 cells</td>
			<td>ATCC</td>
			<td>CCL-247</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CytoTox-Fluo Cytotoxicity Assay</td>
			<td>Promega Corp.</td>
			<td>G9260</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>MultiFlo Microplate Dispenser</td>
			<td>Labsystems Inc.</td>
			<td>Model 832</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Biomek FXP Laboratory Automation Workstation</td>
			<td>Beckman Coulter Inc.</td>
			<td>A31842</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PHERAstar FS-multi-mode HTS microplate reader</td>
			<td>BMG Labtech Inc.</td>
			<td>0471-101A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bright-Line Reichert Hemacytometers</td>
			<td>Hausser Scientific Company</td>
			<td>1490</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

a multiplexed luciferase-based screening platform for interrogating cancer-associated signal transduction in cultured cells

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.

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/&beta;-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...

Watch this Scientific Journal Video about A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells at JoVE.com

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

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  ...

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11.0K vistas.  UT Southwestern Medical Center. El logro de un nivel de comprensión de los sistemas de procesos celulares es un objetivo de la biología celular moderna. Se describe aquí estrategias para luciferase reporteros multiplexación de varias funciones celulares puntos finales para interrogar la función de genes utilizando RNAi bibliotecas escala del genoma.