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: 8548039
• 负责人: MARNIE E HALPERN
• 金额: $ 32.82万
• 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/8548039
• 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（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。在透明的斑马鱼胚胎中，没有什么比这更明显了，在透明的斑马鱼胚胎中，从诸如编码荧光蛋白的转基因的表达允许分离的细胞或组织组的分离组或随着时间的推移，并随着时间的推移，并具有适当的调节剂，可以进行基因修饰或靶向销毁。二进制转录调节系统（例如酵母的GAL4/UAS系统）通过实现基因激活的精确时间和空间控制，彻底改变了果蝇的研究。但是，该系统在斑马鱼和其他脊椎动物模型中的有效性较低，这在很大程度上是由于促进综合转基因的高水平基因表达所需的多拷贝上游激活序列（UAS）的沉默。 在这个项目中，我们将适应用于转基因斑马鱼的Neurospora Crassa的Q调节系统。 Q系统具有许多优势，包括1。比Gal4，2。的转录激活水平更高。转录调节器QF，QF，QF可以被压抑剂QS，QS，3，阻止抑制和恢复QF活性的能力，在QU含量的情况下，QF cp din（quas dient），din。斑马鱼中的甲基化和转录沉默。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。我们建议进一步验证斑马鱼中Q系统的组成部分，以开发新方法用于交叉基因表达，以在Gateway兼容的载体中生成所有试剂，以易于其他研究人员使用，并执行大型增强器陷阱屏幕。我们旨在建立一个集合QF驱动线，以独特的组织特异性模式激活报告基因。特别感兴趣的是对神经增强子陷阱的识别，这些陷阱将在未来的有关大脑发育和行为的研究中具有很高的价值。