Dahlgaard Katja, Troelsen Jesper Thorvald
Department of Science and Environment, Roskilde University, Roskilde, Denmark.
Methods Mol Biol. 2018;1765:57-77. doi: 10.1007/978-1-4939-7765-9_4.
Several tumor suppressors possess gene regulatory activity. Here, we describe how promoter and promoter/enhancer reporter assays can be used to characterize a colorectal tumor suppressor proteins' gene regulatory activity of possible target genes. In the first part, a bioinformatic approach to identify relevant gene regulatory regions of potential target genes is presented. In the second part, it is demonstrated how to prepare and carry out the functional assay.We explain how to clone the bioinformatically identified gene regulatory regions into luciferase reporter plasmids by the use of the quick and efficient In-Fusion cloning method, and how to carry out transient transfections of Caco-2 colon cancer cells with the produced luciferase reporter plasmids using polyethyleneimine (PEI). A plan describing how to set up and carry out the luciferase expression assay is presented. The luciferase/β-galactosidase (Dual Light) assay presented is a highly sensitive assay that can monitor small changes in the promoter/enhancer activity and includes an internal control monitoring transfection efficiency.
几种肿瘤抑制因子具有基因调控活性。在此，我们描述了如何使用启动子和启动子/增强子报告基因检测来表征结直肠癌肿瘤抑制蛋白对可能的靶基因的基因调控活性。在第一部分，介绍了一种通过生物信息学方法来识别潜在靶基因的相关基因调控区域。在第二部分，展示了如何准备和进行功能检测。我们解释了如何使用快速高效的In-Fusion克隆方法将通过生物信息学鉴定的基因调控区域克隆到荧光素酶报告质粒中，以及如何使用聚乙烯亚胺（PEI）对产生的荧光素酶报告质粒进行Caco-2结肠癌细胞的瞬时转染。还给出了一个关于如何设置和进行荧光素酶表达检测的方案。所介绍的荧光素酶/β-半乳糖苷酶（双荧光）检测是一种高度灵敏的检测方法，可监测启动子/增强子活性的微小变化，并包括一个监测转染效率的内部对照。
