Traditional methods to interrogate promoter and protein function use exogenous plasmid based overexpression or surrogate markers of activity and can result in artefactual data. NanoLuc is a novel, small and bright luciferase from the deep sea shrimp Oplophorus gracilirostris and developed by Promega. When compared to traditional luciferases, such as firefly (Photinus pyralis) or Renilla reniformis, NanoLuc is approximately 150 fold brighter and can be detected even at low expression levels. This enables detection of endogenous gene transcription and protein expression. The brightness of NanoLuc combined with its small size, high thermal stability, activity over a broad pH range, lack of post-translational modifications and uniform cellular distribution make it an ideal tool for studying genes or proteins of interest.
In collaboration with Promega, Horizon Discovery has developed a series of NanoLuc® reporter cell lines, which are ideal to study the regulation of endogenous gene and protein expression in a rapid, simple assay system, suitable for use on high throughput platforms.
NanoLuc-PEST promoter reporter cell lines are designed to be used in transcriptional reporter assays. The PEST sequence is a short signal peptide found in proteins with a short intracellular half-life and is involved in targeting proteins for degradation. The inclusion of a PEST sequence in the reporter cell lines reduces the half-life of NanoLuc, ensures a good dynamic range and prevents intracellular accumulation.
NanoLuc-PEST luciferase reporter cell lines allow transcriptional reporter output of endogenous promoter/response elements that drive the NanoLuc expression. The NanoLuc-PEST sequence is placed directly downstream of the start codon of the gene of interest, allowing assessment of endogenous promoter activity.
Dynamics of NanoLuc-PEST promoter reporter lines. Three NanoLuc-PEST Promoter reporter lines for HIF1A, MYC and GLI1 were treated with actinomycin D, a general transcriptional inhibitor, for 6hrs. Rapid and robust decreases in luciferase signal were observed. The data shows good inducible kinetics on inducible genes. CellTiter-Blue® was used to control for effects on cell viability.
NanoLuc protein reporter cell lines are designed to generate a fusion of NanoLuc to the protein of interest and allow interrogation of protein turnover and stability. The fusion is either N or C-terminal and is designed to ensure protein biology is not compromised.
In C-terminal fusions, NanoLuc is introduced directly upstream of the endogenous stop codon in the gene of interest, in N-terminal fusions it is introduced directly downstream of the endogenous start codon. When fused to an endogenous protein of interest, NanoLuc half-life is then linked to the turnover of the protein.
Dynamics of protein reporter cell lines. Three NanoLuc-PEST Promoter reporter lines for HIF1A and NFE2L2 were treated with cyclophosphamide (t1/2 dictated by target genes) to measure turnover and bortezomib, a proteasome inhibitor, for accumulation (4hr treatment). Good repression and accumulation kinetics of signals were observed.
Protein Reporter Control cell lines may also be available. NanoLuc has a long intracellular half-life of ~120minutes and can therefore accumulate in cells. These cell lines are intended as controls for the protein reporter cell lines and are not designed for use in transcriptional reporter assays. This configuration enables researchers to demonstrate that the changes in the luciferase signal are due to changes in the level of the target protein and not the turnover of NanoLuc protein.
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