Leung Chi K, Deonarine Andrew, Strange Kevin, Choe Keith P
Department of Biology, University of Florida, FL, USA.
J Vis Exp. 2011 May 19(51):2745. doi: 10.3791/2745.
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).
高通量筛选（HTS）是一种用于识别生物过程化学调节剂的强大方法。然而，在使用细胞培养模型进行的筛选中鉴定出的许多化合物，在体内往往被发现具有毒性或药理活性不足（1 - 2）。在全动物模型中进行筛选有助于避免这些缺陷，并简化药物开发的流程。秀丽隐杆线虫是一种非常适合高通量筛选的多细胞模式生物。它体型小（<1毫米），可以经济地在液体中培养和分配。秀丽隐杆线虫也是实验上最易于操作的动物模型之一，能够快速且详细地鉴定药物作用模式（3）。我们描述了一种用于培养和分配秀丽隐杆线虫荧光菌株的方案，用于高通量筛选化学文库或检测改变特定基因表达的环境污染物。大量发育同步的线虫在液体培养中生长，收获、洗涤后，以确定的密度悬浮。然后使用蠕动式液体分配器将线虫添加到黑色平底384孔板中。可以将来自化学文库的小分子或测试样品（例如水、食物或土壤）添加到有线虫的孔中。在体内，使用荧光酶标仪测量实时荧光强度。该方法可以适用于秀丽隐杆线虫中任何有合适报告基因的可诱导基因。秀丽隐杆线虫中许多可诱导的应激和发育转录途径已被明确界定，并且已经存在许多针对这些途径的绿色荧光蛋白转基因报告菌株（4）。当与适当的转基因报告基因结合使用时，我们的方法可用于筛选途径调节剂或开发用于检测环境污染物的强大生物传感器检测方法。我们通过我们开发的一种用于监测秀丽隐杆线虫帽状衣领转录因子SKN - 1的高通量筛选试验，展示了我们的秀丽隐杆线虫培养和分配方案。SKN - 1及其哺乳动物同源物Nrf2在氧化应激和异生物应激期间激活细胞保护基因（5 - 10）。Nrf2保护哺乳动物免受许多与年龄相关的疾病，如癌症、神经退行性变和慢性炎症，并且已成为一个主要的化疗靶点（11 - 13）。我们的试验基于SKN - 1靶基因gst - 4的绿色荧光蛋白转基因报告基因（14），该基因编码一种谷胱甘肽 - S转移酶（6）。gst - 4报告基因也是一种用于激活SKN - 1的异生物和氧化化学物质的生物传感器，可用于检测低水平的污染物，如丙烯酰胺和甲基汞（15 - 16）。
