Shimoda Marine Research Center, University of Tsukuba, Shimoda, Shizuoka 415-0025, Japan.
Dev Biol. 2013 Mar 1;375(1):79-91. doi: 10.1016/j.ydbio.2012.12.012. Epub 2012 Dec 27.
Enhancers are flexible in terms of their location and orientation relative to the genes they regulate. However, little is known about whether the flexibility can be applied in every combination of enhancers and genes. Enhancer detection with transposable elements is a powerful method to identify enhancers in the genome and to create marker lines expressing fluorescent proteins in a tissue-specific manner. In the chordate Ciona intestinalis, this method has been established with a Tc1/mariner superfamily transposon Minos. Previously, we created the enhancer detection line E[MiTSAdTPOG]15 (E15) that specifically expresses green fluorescent protein (GFP) in the central nervous system (CNS) after metamorphosis. In this study, we identified the causal insertion site of the transgenic line. There are two genes flanking the causal insertion of the E15 line, and the genomic region around the insertion site contains the enhancers responsible for the expression in the endostyle and gut in addition to the CNS. We found that the endostyle and gut enhancers show sensitivity to the orientation of the GFP gene for their enhancer activity. Namely, the enhancers cannot enhance the expression of GFP which is inserted at the same orientation as the E15 line, while the enhancers can enhance GFP expression inserted at the opposite orientation. The CNS enhancer can enhance GFP expression in both orientations. The DNA element adjacent to the endostyle enhancer is responsible for the orientation sensitivity of the enhancer. The different sensitivity of the enhancers to the orientation of the transgene is a cause of CNS-specific GFP expression in the E15 line.
增强子在其相对于所调节基因的位置和方向上具有灵活性。然而，关于这种灵活性是否可以应用于增强子和基因的每一种组合，人们知之甚少。利用转座元件检测增强子是一种在基因组中识别增强子并创建以组织特异性方式表达荧光蛋白的标记线的强大方法。在脊索动物 Ciona intestinalis 中，已经使用 Tc1/mariner 超家族转座子 Minos 建立了这种方法。此前，我们创建了增强子检测系 E[MiTSAdTPOG]15（E15），该系在变态后特异性地在中枢神经系统（CNS）中表达绿色荧光蛋白（GFP）。在这项研究中，我们确定了转基因系的因果插入位点。E15 系的因果插入有两个基因侧翼，插入位点周围的基因组区域除了 CNS 外，还包含负责在内胚层和肠道表达的增强子。我们发现，内胚层和肠道增强子对其增强子活性的 GFP 基因取向表现出敏感性。即，这些增强子不能增强与 E15 系相同取向插入的 GFP 的表达，而增强子可以增强相反取向插入的 GFP 表达。CNS 增强子可以增强两种取向 GFP 的表达。紧邻内胚层增强子的 DNA 元件负责增强子的取向敏感性。增强子对转基因取向的不同敏感性是 E15 系 CNS 特异性 GFP 表达的原因。
