उच्च throughput और प्रतिदीप्त के साथ स्क्रीनिंग biosensing सी. एलिगेंस खींच
Summary
तरल आधारित संवर्धन और वितरण के लिए एक प्रक्रिया<em> सी. एलिगेंस</em> फ्लोरोसेंट संवाददाता प्रोटीन व्यक्त उपभेदों में वर्णित है कि महंगी छँटाई उपकरण की आवश्यकता नहीं है. यह दृष्टिकोण कई inducible करने के लिए लागू किया जा सकता है<em> सी. एलिगेंस</em> दवा या contaminants की खोज biosensing के लिए जीन.
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 vivo1-2. 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-action3.
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 them4. 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 stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. 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-mercury15-16.
Protocol
Discussion
हम संवर्धन के लिए एक तरीका मौजूद है और ट्रांसजेनिक नेमाटोड के बड़ी संख्या में वितरण. कीड़े संवर्धन के लिए उपकरणों का इस्तेमाल किया आणविक क्लोनिंग प्रदर्शन प्रयोगशालाओं के लिए मानक है और तरल से निपटने…
Disclosures
The authors have nothing to disclose.
Acknowledgements
सी एलिगेंस transgenes Caenorhabditis जेनेटिक्स केंद्र (मिनेसोटा, Minneapolis के विश्वविद्यालय, MN) द्वारा प्रदान किया गया. यह काम एनआईएच R21 केएस NS0667678 – 01 अनुदान द्वारा समर्थित किया गया. सभी लेखकों संग्रह, विश्लेषण, और डेटा की व्याख्या में भाग लिया. सीकेएल, के एस, और KPC लेखन और पांडुलिपि में संशोधन में भाग लिया.
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
|---|---|---|---|
| LB broth | Research Products International Corp. | L24066 | |
| Terrific broth | Research Products International Corp. | T15100 | |
| Synergy™ HT Multi-mode Microplate Reader | BioTek | Filters: GFP 485/20ex 528/20em; RFP 540/25ex 590/35em | |
| MicroFlo Select Dispenser | BioTek | ||
| Worm dispensing flask | Southern Scientific Inc., Micanopy, FL | Custom assembled (352) 284-2531 | |
| 384 microplates | Greiner Bio-One | 5678-1209 | |
| Breathable sealing tape | Nunc | 241205 | |
| (5-hydroxy-p-naphthoqinone) Juglone | ACROS | 121640010 | Juglone is dissolved in DMSO |
| DMSO | Sigma-Aldrich | D-5879 | |
| C. elegans transgenic strain | Author’s laboratory | VP596 | Pgst-4::GFP and Pdop-3::RFP |
References
- Simeonov, A. Quantitative high-throughput screen identifies inhibitors of the Schistosoma mansoni redox cascade. PLoS Negl Trop Dis. 2, e127-e127 (2008).
- Giacomotto, J., Segalat, L. High-throughput screening and small animal models, where are we. Br J Pharmacol. 160, 204-216 (2010).
- Artal-Sanz, M., de Jong, L., Tavernarakis, N. Caenorhabditis elegans: a versatile platform for drug discovery. Biotechnol J. 1, 1405-1418 (2006).
- An, J. H., Blackwell, T. K. SKN-1 links C. elegans mesendodermal specification to a conserved oxidative stress response. Genes Dev. 17, 1882-1893 (2003).
- Choe, K., Przybysz, A., Strange, K. The WD40 repeat protein WDR-23 functions with the CUL4/DDB1 ubiquitin ligase to regulate nuclear abundance and activity of SKN-1 in Caenorhabditis elegans. Mol Cell Biol. 29, 2704-2715 (2009).
- Hasegawa, K. Acrylamide-responsive genes in the nematode Caenorhabditis elegans. Toxicol. Sci. 101, 215-225 (2008).
- Tullet, J. M. A. Direct inhibition of the longevity-promoting factor SKN-1 by insulin-like signaling in C. elegans. Cell. 132, 1025-1038 (2008).
- Oliveira, R. P. Condition-adapted stress and longevity gene regulation by Caenorhabditis elegans SKN-1/Nrf. Aging Cell. , (2009).
- Park, S. -. K., Tedesco, P. M., Johnson, T. E. Oxidative stress and longevity in Caenorhabditis elegans as mediated by SKN-1. Aging Cell. 8, 258-269 (2009).
- Hayes, J. D., McMahon, M., Chowdhry, S., Dinkova-Kostova, A. T. Cancer chemoprevention mechanisms mediated through the Keap1-Nrf2 pathway. Antioxid Redox Signal. , (2010).
- Kwak, M. K., Kensler, T. W. Targeting NRF2 signaling for cancer chemoprevention. Toxicol Appl Pharmacol. 244, 66-76 (2010).
- Leiser, S. F., Miller, R. A. Nrf2 signaling, a mechanism for cellular stress resistance in long-lived mice. Mol Cell Biol. 30, 871-884 (2010).
- Link, C. D., Johnson, C. J. Reporter transgenes for study of oxidant stress in Caenorhabditis elegans. Methods Enzymol. 353, 497-505 (2002).
- Vanduyn, N., Settivari, R., Wong, G., Nass, R. SKN-1/Nrf2 inhibits dopamine neuron degeneration in a Caenorhabditis elegans model of methylmercury toxicity. Toxicol Sci. , (2010).
- Hasegawa, K. A rapid and inexpensive method to screen for common foods that reduce the action of acrylamide, a harmful substance in food. Toxicol Lett. 175, 82-88 (2007).
- Chase, D. L., Pepper, J. S., Koelle, M. R. Mechanism of extrasynaptic dopamine signaling in Caenorhabditis elegans. Nature Neuroscience. 7, 1096-1103 (2004).
- Hope, I. A. . C. elegans, a Practical Guide. , (2005).
- Pulak, R. Techniques for analysis, sorting, and dispensing of C. elegans on the COPAS flow-sorting system. Methods Mol Biol. 351, 275-286 (2006).
