Geissler Rene, Grimson Andrew
Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA.
Methods Mol Biol. 2018;1720:77-88. doi: 10.1007/978-1-4939-7540-2_6.
GFP reporter constructs are widely used as an expression system for studying the function of regulatory sequence motifs (cis elements) within the 3'-UTRs (3' untranslated regions) of mRNAs. Here we provide details on the characterization of individual sequence motifs, which typically regulate mRNA decay and translation. In addition, we describe methods to identify trans factors required for the function of such elements. To facilitate efficient identification of novel functional 3'-UTR motifs, we describe a screening approach based on dual-color fluorescence reporter constructs. Such screening approaches can be used to test large collections of defined sequence or libraries of random sequences.
绿色荧光蛋白（GFP）报告基因构建体被广泛用作一种表达系统，用于研究mRNA的3'非翻译区（3'-UTR）内调控序列基序（顺式元件）的功能。在此，我们提供了关于单个序列基序特征的详细信息，这些基序通常调控mRNA的降解和翻译。此外，我们描述了鉴定此类元件功能所需反式因子的方法。为便于高效鉴定新型功能性3'-UTR基序，我们描述了一种基于双色荧光报告基因构建体的筛选方法。此类筛选方法可用于测试大量定义序列或随机序列文库。
