School of Clinical and Experimental Medicine, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, B15 2TT, UK.
Development. 2014 Feb;141(3):715-24. doi: 10.1242/dev.100347.
Zebrafish transgenesis is increasingly popular owing to the optical transparency and external development of embryos, which provide a scalable vertebrate model for in vivo experimentation. The ability to express transgenes in a tightly controlled spatio-temporal pattern is an important prerequisite for exploitation of zebrafish in a wide range of biomedical applications. However, conventional transgenesis methods are plagued by position effects: the regulatory environment of genomic integration sites leads to variation of expression patterns of transgenes driven by engineered cis-regulatory modules. This limitation represents a bottleneck when studying the precise function of cis-regulatory modules and their subtle variants or when various effector proteins are to be expressed for labelling and manipulation of defined sets of cells. Here, we provide evidence for the efficient elimination of variability of position effects by developing a PhiC31 integrase-based targeting method. To detect targeted integration events, a simple phenotype scoring of colour change in the lens of larvae is used. We compared PhiC31-based integration and Tol2 transgenesis in the analysis of the activity of a novel conserved enhancer from the developmentally regulated neural-specific esrrga gene. Reporter expression was highly variable among independent lines generated with Tol2, whereas all lines generated with PhiC31 into a single integration site displayed nearly identical, enhancer-specific reporter expression in brain nuclei. Moreover, we demonstrate that a modified integrase system can also be used for the detection of enhancer activity in transient transgenesis. These results demonstrate the power of the PhiC31-based transgene integration for the annotation and fine analysis of transcriptional regulatory elements and it promises to be a generally desirable tool for a range of applications, which rely on highly reproducible patterns of transgene activity in zebrafish.
斑马鱼转基因技术因其胚胎的光学透明性和外部发育而越来越受欢迎，为体内实验提供了可扩展的脊椎动物模型。在严格控制的时空模式下表达转基因的能力是在广泛的生物医学应用中利用斑马鱼的重要前提。然而，传统的转基因方法受到位置效应的困扰：基因组整合位点的调节环境导致工程化顺式调控模块驱动的转基因表达模式的变化。当研究顺式调控模块及其细微变体的精确功能或表达各种效应蛋白以标记和操作定义的细胞集时，这种限制代表了一个瓶颈。在这里，我们通过开发基于 PhiC31 整合酶的靶向方法提供了消除位置效应可变性的证据。为了检测靶向整合事件，使用幼虫晶状体颜色变化的简单表型评分来检测。我们比较了基于 PhiC31 的整合和 Tol2 转基因在分析发育调节的神经特异性 esrrga 基因的新型保守增强子活性中的作用。Tol2 生成的独立系中报告基因的表达高度可变，而在单个整合位点中用 PhiC31 生成的所有系中，报告基因在脑核中均表现出几乎相同的增强子特异性表达。此外，我们证明了改良的整合酶系统也可用于瞬态转基因中增强子活性的检测。这些结果证明了基于 PhiC31 的转基因整合在转录调控元件的注释和精细分析中的强大功能，并且有望成为一系列应用的理想工具，这些应用依赖于斑马鱼中转基因活性的高度可重复模式。
