Reporter gene assays are widely used to study the regulation of gene expression. We have developed a suite of endogenous reporter cell lines which measure natural levels of protein expression and promoter activity. By measuring at the endogenous level, this system provides an advantage over other technologies which use exogenous plasmid-based overexpression systems.
Cell line development and validation
Using adeno-associated virus based gene editing HCT116 cells were engineered at the endogenous HIF1A locus to generate an in-frame NanoLuc® luciferase protein fusion. NanoLuc® luciferase is a small enzyme engineered for optimal performance as a luminescent reporter. The bright nature of NanoLuc® luciferase enables activity to be detected at endogenous expression levels. The below figure highlights the relative luminescence levels of the different luciferases, as well as the signal range we observe from the various endogenous reporter cell lines we have developed.
Validation experiments were first performed to assess the utility of the HIF1A NanoLuc® protein reporter in drug discovery and development processes. Protein turnover was detected by using cycloheximide (A) and protein accumulation was measured using bortezomib (B) to block protein degradation. Furthermore, it was demonstrated that a linear relationship between luciferase signal and cell number is maintained even at low cell numbers (C), and decay kinetics showed a stable signal half-life (B).
Exposure to hypoxia (1% oxygen) for 4 hours resulted in the stabilization of HIF1A protein leading to an increase in luciferase signal. Treatment with the HIF1A pathway inhibitor YC-1 decreased expression of HIF1A under hypoxia, as determined by NanoLuc® protein reporter.
These results were confirmed by Western blotting for HIF1A protein.
High-Throughput Screen (HTS) with HIF1A Endogenous Reporter Line
Cells were seeded into 1536 -well plates and induction of HIF1A performed using 1% oxygen or the hypoxic-mimetic CoCl2. Knockdown of HIF1A protein reporter signal was confirmed using Topotecan. A high-throughput screen was run in 1536-well plate format using the NCGC Pharmaceutical Collection of approved and investigational drugs. The assay performed well with Z’ of 0.70 and CV of 6.1%.
Initial results following a 16 hour drug exposure identified known regulators of the HIF1A pathway, highlighting the utility of this system for high-throughput screening.
We have generated novel protein reporter cell lines by introducing NanoLuc®, allowing us to study the modulation of endogenous protein stability in the high-throughput setting.This technology could be applied to virtually any gene of interest and in a range of cell backgrounds, with resultant cell lines having applications in basic research as well as in high-throughput compound library screens