A gene-trap screen for hearing and balance

用于听力和平衡的基因陷阱筛查

• 批准号: 7296202
• 负责人: Donna M Fekete
• 金额: $ 21.2万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-15 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7296202
• 关键词: Ablation; Animal Model; Auditory; Auditory system; Behavior; Breeding; Candidate Disease Gene; Cell Lineage; Cell Separation; Cells; Cellular Mechanotransduction; Complex; Congenital Abnormality; DNA Transposable Elements; Defect; Detection; Development; Diphtheria Toxin; Disease; Dominant-Negative Mutation; Embryo; Equilibrium; Event; Fishes; Funding Mechanisms; Gene Expression Regulation; Gene Targeting; Genes; Genetic; Goals; Hearing; Human; Image; Injection of therapeutic agent; Labyrinth; Lead; Life; Live Birth; Maps; Methods; Modeling; Mutation; Nature; Neuraxis; Neurons; Numbers; Organ; Peripheral; Pilot Projects; Population; Process; Proteins; RNA Splicing; Reporter Genes; Retroviral Vector; Retroviridae; Risk; Role; Sensorineural Hearing Loss; Site; System; Testing; Tissues; Toxin; Transgenic Organisms; Transposase; Zebrafish; base; congenital deafness; deafness; design; fluorescence imaging; gain of function; gene discovery; hearing impairment; in vivo; killings; loss of function; mature animal; mutant; novel; promoter; protein expression; relating to nervous system; response; vector

DESCRIPTION (provided by applicant): Congenital forms of sensorineural hearing loss can arise from perturbations in development of either peripheral- or central-nervous-system components. Gene discovery approaches that can identify new genes expressed during development of the auditory or vestibular systems in animal models should assist in revealing genetic causes of congenital deafness in humans. We have devised a new Gal4-UAS-based gene- trap screen for zebrafish embryos that should facilitate not only gene discovery, but also both loss-of-function and gain-of-function approaches for testing candidate genes involved in complex processes such as development of the auditory and vestibular systems. Furthermore, our gene-trapping strategy should be applicable to any developing organ or system in the zebrafish, thereby enhancing the versatility of zebrafish as an important model organism for understanding the genetic causes of various human birth defects. We propose two Specific Aims. (1) To generate new otic- or neural-specific gene-trap lines in zebrafish. Pseudotyped retroviral vectors or Tol2 transposases will be used to insert a gene-trap construct into the zebrafish germline. The trapping construct will use a GAL4-UAS system to transactivate the expression of a reporter gene that can be screened by fluorescence imaging of live embryos. Lines showing relatively specific expression in peripheral or central components of mechanosensory systems will be created and trapped genes will be cloned. (2) To use gene-trapped Gal4-driver lines for targeted cell ablation in vivo. One major advantage of our gene-trap design is its potential for targeting bioactive molecules to specific cells in vivo without requiring the isolation of cell- or tissue-specific promoters. This can be accomplished by crossing a particular Gal4-trap line (i.e., the activator line) with a transgenic line carrying a target gene placed downstream of a UAS sequence (i.e., the effector line). Only when both the activator and effector are active in the same cells is the effector protein expressed. An inducible form of Gal4 (GeneSwitch) will permit even more control over the onset of effector protein expression. As proof-of-principle, an effector line will be created with UAS upstream of a toxin gene. When crossed to any of the driver lines, we expect the toxin will specifically kill only those cells expressing the trapped gene. This should prove especially powerful for selective ablation of subsets of CNS neurons to assess their role in development and/or in behavior. Our novel gene-trap screen should facilitate gene discovery in zebrafish, and readily allow tests of candidate genes for their involvement in development of the auditory system. Our long-term goal is to determine whether any of the newly discovered hearing-related genes in zebrafish also correspond to genes underlying congenital deafness in humans.

描述（由申请人提供）：先天性的感官听力损失的形式可能是由于周围或中央导致系统组件的开发而产生的。可以鉴定动物模型中听觉或前庭系统发展过程中表达的新基因的基因发现方法应有助于揭示人类先天性耳聋的遗传原因。我们为斑马鱼胚胎设计了一个新的基于GAL4-UAS的基因筛选，不仅应该促进基因发现，而且还应促进功能丧失和功能障碍方法，用于测试参与复杂过程的候选基因，例如开发听觉和前庭系统。此外，我们的基因捕获策略应适用于斑马鱼中的任何正在发育的器官或系统，从而增强斑马鱼作为理解各种人类先生缺陷的遗传原因的重要模型生物的多功能性。我们提出了两个具体目标。 （1）在斑马鱼中生成新的耳或神经特异性基因陷阱线。伪型逆转录病毒载体或TOL2转座酶将用于将基因陷阱构建体插入斑马鱼种系中。捕获构建体将使用GAL4-UAS系统来反式激活报告基因的表达，该基因可以通过活胚的荧光成像筛选。将创建在机械感觉系统的外围或中央组件中显示相对特异性表达的线，并将克隆基因。 （2）在体内使用受基因捕获的GAL4驱动器线进行靶向细胞消融。我们的基因陷阱设计的一个主要优点是它的潜力是将生物活性分子靶向体内特定细胞，而无需分离细胞或组织特异性启动子。这可以通过将特定的GAL4陷阱线（即激活剂线）与带有UAS序列下游的靶基因的转基因线（即效应线）的转基因线（即激活剂线）来实现。只有当激活因子和效应子在同一细胞中都活跃时，才表达效应蛋白。 GAL4（Geneswitch）的诱导形式将允许对效应蛋白表达的发作有更多的控制。作为原理证明，将使用毒素基因上游创建效应线。当越过任何驱动线时，我们预计毒素将特别杀死那些表达被困基因的细胞。对于选择性消融中枢神经系统神经元子集以评估其在发展和/或行为中的作用，这应该是特别有力的。我们新颖的基因陷阱筛选应促进斑马鱼中的基因发现，并很容易地允许候选基因参与听觉系统的发展。我们的长期目标是确定斑马鱼中的任何新发现的与听力相关的基因是否也对应于人类的先天性耳聋的基因。