Nanoluciferase-Based Method for Detecting Gene Expression in

Genetic reporters such as the green fluorescent protein (GFP) can facilitate measurement of promoter activity and gene expression. However, animal autofluorescence limits the sensitivity of GFP and other fluorescent reporters in whole-animal settings like in the nematode Here, we present a highly sensitive Nanoluciferase (NanoLuc)-based method in a multiwell format to detect constitutive and inducible gene expression in We optimize detection of bioluminescent signals from NanoLuc in and show that it can be detected at 400,000-fold over background in a population of 100 animals expressing intestinal NanoLuc driven by the promoter. We can reliably detect signal in single ::-expressing worms from all developmental stages. Furthermore, we can detect signal from a 1/100 dilution of lysate from a single ::-expressing adult and from a single ::-expressing adult "hidden" in a pool of 5000 N2 wild-type animals. We also optimize various steps of this protocol, which involves a lysis step that can be performed in minutes. As a proof-of-concept, we used NanoLuc to monitor the promoter activity of the stress/immune reporter and were able to measure 300- and 50-fold increased NanoLuc activity after proteasome blockade and infection with microsporidia, respectively. Altogether, these results indicate that NanoLuc provides a highly sensitive genetic reporter for rapidly monitoring whole-animal gene expression in .