Transgenic Tools for Regulated Gene Expression in Zebrafish

描述(由申请人提供)：控制体内基因表达的工具在实验生物学和疾病过程研究中发挥着重要作用。这一点在透明斑马鱼胚胎中表现得最为明显。在透明斑马鱼胚胎中，通过转基因(如编码荧光蛋白的基因)的表达，可以随着时间的推移标记和跟踪不同的细胞或组织群，并在适当的调节剂的作用下，对其进行转基因或靶向销毁。二元转录调控系统，如酵母的Gal4/UAS系统，通过精确地控制基因激活的时间和空间，彻底改变了果蝇的研究。然而，该系统在斑马鱼和其他脊椎动物模型中的有效性较低，这在很大程度上是因为多拷贝上游激活序列(UA)的甲基化和沉默，这是促进整合转基因高水平基因表达所必需的。在本项目中，我们将使粗糙脉孢菌的Q调控系统用于转基因斑马鱼。Q系统有许多优点，包括1.比Gal4实现更高水平的转录激活，2.转录调节因子QF，它可以被抑制物QS失活，3.在奎尼酸存在的情况下阻断抑制并恢复QF活性的能力，以及重要的是，不包含必要的CpG二核苷酸的QF结合位点(QAs)，这在斑马鱼中容易发生DNA甲基化和转录沉默。最近，Q系统被成功地应用于无脊椎动物模型。我们的初步数据表明，它还能有效地调节斑马鱼胚胎的转录。我们建议进一步验证斑马鱼Q系统的组件，开发新的交叉基因表达方法，在Gateway兼容载体中生成所有试剂以便于其他研究人员使用，并进行大规模的增强子陷阱筛选。我们的目标是建立一个能够以独特的组织特异性模式激活报告基因的QF驱动系集合。特别令人感兴趣的是神经增强器陷阱的识别，这将在未来对大脑发育和行为的研究中具有重要价值。

DESCRIPTION (provided by applicant): Tools to control the expression of genes in vivo play an essential role in experimental biology and research on disease processes. Nowhere is this more apparent than in the transparent zebrafish embryo, where expression from transgenes such as those encoding fluorescent proteins allows discrete groups of cells or tissues to be labeled and followed over time, and with appropriate regulators, to be genetically modified or targeted for destruction. Binary transcriptional regulatory systems, such as the Gal4/UAS system of yeast, have revolutionized research in Drosophila by enabling precise temporal and spatial control of gene activation. However, this system has been less effective in zebrafish and other vertebrate models, in large part due to the progressive methylation and silencing of multicopy upstream activation sequences (UAS) needed to promote high levels of gene expression from integrated transgenes. In this project, we will adapt the Q regulatory system of Neurospora crassa for use in transgenic zebrafish. The Q system has many advantages, including 1. a higher level of transcriptional activation than achieved by Gal4, 2. a transcriptionl regulator, QF, that can be inactivated by a repressor, QS, 3, the ability to block repression and restore QF activity in the presence of quinic acid and, importantly, 4. a QF binding site (QUAS) that does not contain essential CpG dinucleotides, which are prone to DNA methylation and transcriptional silencing in zebrafish. Recently, the Q system was successfully applied to invertebrate models. Our preliminary data indicate that it also functions effectively to regulate transcription in zebrafish embryos. We propose to validate further the components of the Q system in zebrafish, to develop new methods for intersectional gene expression, to generate all reagents in Gateway compatible vectors for ease of use by other researchers, and to perform a large-scale, enhancer trap screen. We aim to establish a collection QF driver lines that activate reporter genes in unique, tissue-specific patterns. Of special interest is the identification of neural enhancer traps that will be of great value in future studies on brain development and behavior.