Abstract 5522: X-MAN™ reporter cell lines: Enabling the study of endogenous promoter activity and protein dynamics.
Authors: Astley Holly , Grooby Suzanne , Francis Jo , Griffin Sue , Little Annette , Benink Hélène , Kelly Jeff , Foster Rebecca 
Published: August 2014

Abstract
To allow promoter activity and protein dynamics to be studied at endogenous levels for the first time, Horizon Discovery has developed suites of X-MAN™ reporter cell lines incorporating NanoLuc™ and HaloTag® technologies from Promega. By creating these innovative reporters, we have removed the need for either exogenous plasmid based overexpression studies, or use of surrogate markers of activity, both of which can yield artefactual data. Horizon's proprietary rAAV-based GENESIS™ gene editing platform has been used to introduce reporter genes (NanoLuc™ or HaloTag®) into several specific chromosomal loci (including HIF1A, cMYC, β-Catenin and NRF2) either as endogenous promoter fusions or in-frame protein fusions. Our extensive expertise in cell line engineering means that the technology can be rapidly applied to virtually any gene of interest. NanoLuc™ produces high intensity luminescence enabling accurate quantification of gene expression even at low endogenous expression levels. HaloTag® is a multifunctional protein reporter which can be used for many applications including intracellular fluorescent imaging of live cells in real time. Validation experiments, including kinetic measurements and treatment with compounds or conditions that modulate transcription or protein expression, reveal robust and reproducible results for all reporter cell lines and demonstrate their value in a wide variety of applications, from pathway analysis to high throughput screening platforms. To further demonstrate the utility of the X-MAN™ reporter cell lines in HTS-screening applications, we used the HCT116 HIF1A NanoLuc™ protein reporter line in a multiplexed siRNA library screen against 960 ‘druggable’ targets, under both normoxic and reduced oxygen conditions. As expected, many known regulators of HIF1A were identified, such as AKT, PDK1 and cRaf, showing once more the robust nature of the reporter system. Several novel regulators were also identified highlighting the value of the reporter cell lines for rapid identification of key regulators of endogenous proteins. In conclusion, we have used the combination of NanoLuc™, HaloTag® and the GENESIS™ gene editing platform to generate highly sensitive reporter cell lines that are capable of registering physiological levels of gene transcription and protein activity/localization in live cells. These reporter technologies can be used for a wide range of applications and provide an exciting new tool for biologically relevant drug discovery.
Citation Format: Holly Astley, Suzanne Grooby, Jo Francis, Sue Griffin, Annette Little, Hélène Benink, Jeff Kelly, Rebecca Foster. X-MAN™ reporter cell lines: Enabling the study of endogenous promoter activity and protein dynamics. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5522. doi:10.1158/1538-7445.AM2013-5522