A gene-trap screen for hearing and balance

描述(申请人提供)：先天性感音神经性听力损失可由外周或中枢神经系统组件的发育障碍引起。基因发现方法可以在动物模型中识别在听觉或前庭系统发育过程中表达的新基因，这将有助于揭示人类先天性耳聋的遗传原因。我们已经为斑马鱼胚胎设计了一种新的基于Gal4-UAS的基因陷阱筛查，它不仅应该有助于基因发现，而且还应该有助于测试参与复杂过程的候选基因，如听觉和前庭系统的发育。此外，我们的基因捕捉策略应适用于斑马鱼任何发育中的器官或系统，从而增强斑马鱼作为了解各种人类出生缺陷的遗传原因的重要模式生物的多样性。我们提出了两个具体目标。(1)在斑马鱼中创造新的听觉或神经特异的基因陷阱系。伪型逆转录病毒载体或Tol2转座酶将被用于将基因陷阱构建插入斑马鱼生殖系。捕获结构将使用GAL4-UAS系统反式激活报告基因的表达，该报告基因可以通过活胚胎的荧光成像进行筛选。将创建在机械感觉系统的外周或中央组件中显示相对特定表达的品系，并克隆捕获的基因。(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将生物活性分子靶向体内特定的细胞，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-陷阱线(即，激活线)与放置在UAS序列下游的携带靶基因的转基因线(即，效应线)杂交来实现。只有当激活剂和效应器在同一细胞中都活跃时，效应器蛋白才能表达。一种可诱导形式的Gal4(GeneSwitch)将允许更多地控制效应器蛋白表达的开始。作为原理证明，将在毒素基因的上游创建一条效应线。当与任何一条驱动线交叉时，我们预计这种毒素只会特异性地杀死那些表达捕获基因的细胞。这应该被证明是特别有效的选择性消融CNS神经元亚群，以评估它们在发育和/或行为中的作用。我们的新基因陷阱筛查应该有助于在斑马鱼中发现基因，并随时可以测试参与听觉系统发育的候选基因。我们的长期目标是确定在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。

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.