Transgenic Tools for Regulated Gene Expression in Zebrafish

用于调节斑马鱼基因表达的转基因工具

• 批准号: 8702212
• 负责人: MARNIE E HALPERN
• 金额: $ 34.26万
• 依托单位: CARNEGIE INSTITUTION OF WASHINGTON, D.C.
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-18 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8702212
• 关键词: Adult; Basic Science; Behavior; Binding; Binding Sites; Biological Assay; Biology; Brain; Caenorhabditis elegans; Cell Lineage; Cells; Collection; Communities; CpG dinucleotide; Cultured Cells; DNA Methylation; Data; Development; Developmental Process; Disease; Drosophila genus; Effectiveness; Embryo; Enhancers; Future; Gene Activation; Gene Expression; Generations; Genes; Genetic Transcription; Genome; Hereditary Disease; Invertebrates; Label; Laboratories; Measures; Methods; Methylation; Modeling; Monitor; Nervous system structure; Neurons; Neurospora crassa; Pattern; Physiological Processes; Play; Process; Property; Proteins; Quinic Acid; Reagent; Reporter Genes; Repression; Research; Research Personnel; Resistance; Role; Solutions; Stem cells; System; Techniques; Testing; Time; Tissues; Transcriptional Activation; Transcriptional Regulation; Transgenes; Transgenic Organisms; Variant; Work; Yeasts; Zebrafish; base; cell behavior; gene function; improved; in vivo; interest; mutant; public health relevance; reconstitution; relating to nervous system; research study; response; tool; transcription factor; transgene expression; vector

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.

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起着至关重要的作用。这一点在透明的斑马鱼胚胎中表现得最为明显。在斑马鱼胚胎中，转基因基因（如编码荧光蛋白的基因）的表达可以让不同的细胞或组织群体被标记，并随着时间的推移被跟踪，在适当的调节下，进行基因改造或以破坏为目标。双转录调控系统，如酵母的Gal4/UAS系统，通过精确控制基因激活的时间和空间，已经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效果不佳，这在很大程度上是由于多拷贝上游激活序列（UAS）的逐渐甲基化和沉默，而多拷贝上游激活序列是促进整合转基因高水平基因表达所必需的。本课题拟将粗神经孢子虫的Q调控系统应用于转基因斑马鱼。Q系统有很多优点，包括1。其转录激活水平高于Gal4, 2。一个转录调节因子QF，可以被一个抑制因子QS灭活，3，在奎宁酸存在下阻断抑制和恢复QF活性的能力，重要的是，4。一个不含必需CpG二核苷酸的QF结合位点（QUAS），这在斑马鱼中容易发生DNA甲基化和转录沉默。最近，Q系统成功地应用于无脊椎动物模型。我们的初步数据表明，它也有效地调节斑马鱼胚胎的转录。我们建议在斑马鱼中进一步验证Q系统的组成部分，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，以方便其他研究人员使用，并进行大规模的增强子陷阱筛选。我们的目标是建立一个QF驱动系集合，以独特的组织特异性模式激活报告基因。特别令人感兴趣的是神经增强器陷阱的识别，这将对未来大脑发育和行为的研究具有重要价值。