High-throughput Screening and Biosensing with Fluorescent <em>C. elegans</em> Strains

Abstract

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo [1][2] . Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.C. elegans is a multicellular model organism well suited for HTS. It is small (<1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action 3 .We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them 4 . When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants.We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap 'n' collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress 5-10 . Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target 11-13 .Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-4 14 , which encodes a glutathione-s transferase 6 . The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury [15][16] . Video LinkThe 1. Add 5 ml of saturated E. coli OP50 bacterial culture to 500 ml Terrific broth supplemented with 50 μg/ml streptomycin and grow in a shaking incubator (225 rpm) overnight at 37°C.2. Split the overnight bacterial culture into ten 50 ml tubes and centrifuge bacteria in a refrigerated centrifuge at 2,500 rcf for 20 minutes. 3. Decant off LB broth and resuspend each bacterial pellet in 10 ml of liquid nematode growth media (NGM). Shake horizontally in a floor shaker for 15...

Unique structure and regulation of the nematode detoxification gene regulator, SKN-1: implications to understanding and controlling drug resistance

“…Given that many parasitic nematodes have limited genetic tractability, the development of small molecule modulators of SKN-1 would provide useful tools for studying the function of this transcriptional pathway. Transgenic C. elegans reporter strains for SKN-1 activity have been developed and perform well in whole-animal high-throughput assays making screening for small-molecule inhibitors feasible (Leung et al 2011). …”

Biochemical and High Throughput Microscopic Assessment of Fat Mass in <em>Caenorhabditis Elegans</em>

“…Moreover, as computational programs become more advanced, there is the prospect for single worm analysis, generating statistically significant results from a single well. Thus our method that utilizes high-throughput microscopy has some advantages over published screen methodologies that use a Copas Biosort to quantify fluorescent signals 32,33 . However, the methodologies are definitely complimentary.…”

SKN-1-independent transcriptional activation of glutathione S-transferase 4 (GST-4) by EGF signaling

“…7,11,12,15,19,20,23,[26][27][28][29][30][31][32][33] A problem arises, however, when GST-4 is used as the sole readout for SKN-1 activation, in the absence of aforementioned controls. 21,22,24,25 Based on our current observations, the interpretation of gst-4 p ::gfp fluorescence in vivo is indeed not straightforward.…”

“…3 Currently, SKN-1 transcriptional activity is often indirectly assayed via a transcriptional reporter construct of the gst-4 promotor (gst-4 p ::gfp or Pgst-4::gfp; strain CL2166) which shows increased GFP levels when transcription is activated in vivo. 7,11,12,15,[19][20][21][22][23][24][25][26][27][28][29][30] By using gst-4 transcription levels as a proxy for SKN-1 activation, the problem of a low GFP signal is mitigated. Additionally, it is possible to obtain a quantitative indication of the gene's transcription levels, instead of merely being able to discriminate between cytoplasmic or nuclear localization of a transcription factor.…”