Nanoluciferase-based Method for Detecting Gene Expression in C. elegans

Abstract

Genetic reporters such as the green fluorescent protein (GFP) can facilitate measurement of promoter activity and gene expression. However, GFP and other fluorophores have limited sensitivity, particularly in tissues that autofluoresce like the intestine of the nematode Caenorhabditis elegans. Here, we present a highly sensitive Nanoluciferase (NanoLuc)-based method in multi-well format to detect constitutive and inducible gene expression in C. elegans. We optimize detection of bioluminescent signal from NanoLuc in C. elegans 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 vha-6 promoter. We can reliably detect signal in single vha-6p::Nanoluc-expressing worms from all developmental stages. Furthermore, we can detect signal from 1/100 dilution of lysate from a single vha-6p::Nanoluc-expressing adult and from a single vha-6p::Nanoluc-expressing adult \"hidden\" in a pool of 5,000 N2 wild-type animals. We also optimized 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 promoter activity of the pals-5 stress/immune reporter and we 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 gene expression in C. elegans.