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: 7448568
• 负责人: Donna M Fekete
• 金额: $ 18.48万
• 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 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7448568
• 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）：先天性感音神经性听力损失可由周围或中枢神经系统组成部分的发育紊乱引起。基因发现方法可以识别动物模型中听觉或前庭系统发育过程中表达的新基因，有助于揭示人类先天性耳聋的遗传原因。我们设计了一种新的基于gal4 - uas的斑马鱼胚胎基因陷阱筛选方法，不仅可以促进基因发现，还可以促进功能丧失和功能获得的方法，用于测试涉及听觉和前庭系统发育等复杂过程的候选基因。此外，我们的基因捕获策略应该适用于斑马鱼的任何发育器官或系统，从而增强斑马鱼作为理解各种人类出生缺陷遗传原因的重要模式生物的多功能性。我们提出两个具体目标。(1)在斑马鱼中产生新的听觉或神经特异性基因诱捕系。将使用假型逆转录病毒载体或Tol2转座酶将基因诱捕结构插入斑马鱼种系。捕获结构将使用GAL4-UAS系统反激活一个报告基因的表达，该报告基因可以通过活胚胎的荧光成像进行筛选。在机械感觉系统的外围或中心成分中表现出相对特定表达的细胞系将被创造出来，被捕获的基因将被克隆。(2)利用基因捕获的gal4驱动系进行体内靶向细胞消融。我们的基因陷阱设计的一个主要优势是它有可能在体内将生物活性分子靶向特定细胞，而不需要分离细胞或组织特异性启动子。这可以通过将特定的gal4诱捕株系（即激活株系）与放置在UAS序列下游的携带靶基因的转基因株系（即效应株系）杂交来实现。只有当激活因子和效应因子在同一细胞中都具有活性时，效应蛋白才会表达。诱导形式的Gal4 （GeneSwitch）将允许更多地控制效应蛋白表达的开始。作为原理证明，将在毒素基因的上游用无人机创建一条效应线。当与任何驱动系杂交时，我们预计毒素只会特异性地杀死那些表达被困基因的细胞。这对于选择性消融中枢神经系统神经元亚群以评估其在发育和/或行为中的作用尤其有效。我们的新基因陷阱筛选将有助于在斑马鱼中发现基因，并很容易地对参与听觉系统发育的候选基因进行测试。我们的长期目标是确定斑马鱼中新发现的与听力相关的基因是否也与人类先天性耳聋的基因相对应。