High-throughput Screening and Biosensing with Fluorescent C-elegans Strains
High-throughput Screening and Biosensing with Fluorescent C-elegans Strains
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DOI：
10.3791/2745
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发表时间：
2011-05-01
影响因子：
1.2
通讯作者：
Choe, Keith P.
中科院分区：
文献类型：
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作者：
Leung, Chi K.;Deonarine, Andrew;Choe, Keith P.
高通量筛选（HTS）是识别生物过程化学调节剂的有力方法。然而，使用细胞培养模型在筛选中发现的许多化合物通常在体内是有毒的或无药理活性的（1-2）。在整个动物模型中进行筛选可以帮助避免这些陷阱，并简化药物开发的途径。秀丽隐杆线虫是一种多细胞模式生物，非常适合高温超导。它很小（< 1毫米），可以经济地培养和分配在液体中。秀丽隐杆线虫也是实验上最容易处理的动物模型之一，允许快速和详细地确定药物的作用模式(3)。我们描述了一种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。大量发育同步的蠕虫在液体培养中生长，收获，洗涤，并以规定的密度悬浮。然后使用蠕动液体分配器将蠕虫添加到黑色的384孔平板上。从化学库或测试样品（如水、食物或土壤）中提取的小分子可以添加到有蠕虫的井中。在体内，实时荧光强度用荧光微孔板读取器测量。该方法适用于秀丽隐杆线虫中任何可诱导基因，只要有合适的报告基因可用。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定义，其中许多已经存在转基因GFP报告菌株(4)。当与适当的转基因报告结合时，我们的方法可用于筛选途径调节剂或开发健壮的生物传感器检测环境污染物。我们展示了秀丽隐杆线虫的培养和分配方案，我们开发了一种HTS检测方法来监测秀丽隐杆线虫帽‘n’领转录因子SKN-1。SKN-1及其哺乳动物同源物Nrf2在氧化和外源应激时激活细胞保护基因（5-10）。Nrf2保护哺乳动物免受许多与年龄相关的疾病，如癌症、神经变性和慢性炎症，并已成为一个主要的化疗靶点（11-13）。我们的实验是基于SKN-1靶基因gst-4（14）的GFP转基因报告基因，该基因编码谷胱甘肽转移酶(6)。gst-4报告基因也是激活SKN-1的异种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）。
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).