Rapid and Efficient Synthetic Assembly of Multiplex Luciferase Reporter Plasmids for the Simultaneous Monitoring of Up to Six Cellular Signaling Pathways.

Current Protocols in Molecular Biology
Alejandro Sarrion-PerdigonesKoen J T Venken

Abstract

High-throughput cell-based screening assays are valuable tools in the discovery of chemical probes and therapeutic agents. Such assays are designed to examine the effects of small compounds on targets, pathways, or phenotypes participating in normal and disease processes. While most cell-based assays measure single quantities, multiplexed assays seek to address these limitations by obtaining multiple simultaneous measurements. The signals from such measurements should be independently detectable and cover large dynamic ranges. Luciferases are good candidates for generation of such signals. They are genetically encoded, versatile, and cost-effective, and their output signals can be sensitively detected. We recently developed a multiplex luciferase assay that allows monitoring the activity of five experimental pathways against one control simultaneously. We used synthetic assembly cloning to assemble all six luciferase reporter units into a single vector over eight stitching rounds. Because all six reporters are on a single piece of DNA, a single vector ensures stoichiometric ratios of each transcriptional unit in each transfected cell, resulting in lower experimental variation. Our proof-of-concept multiplex hextuple luciferase ...Continue Reading

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Jun 17, 2020·Current Protocols in Molecular Biology·Alejandro Sarrion-PerdigonesKoen J T Venken

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