A gene-trap screen for hearing and balance

描述（由申请人提供）：先天性感音神经性听力损失可由外周或中枢神经系统成分发育中的扰动引起。基因发现的方法，可以确定新的基因表达过程中的听觉或前庭系统的动物模型的发展应有助于揭示遗传原因的先天性耳聋在人类。我们已经设计了一种新的基于Gal 4-UAS的斑马鱼胚胎基因捕获筛选，该筛选不仅有助于基因发现，而且还有助于功能丧失和功能获得的方法，用于测试参与复杂过程的候选基因，例如听觉和前庭系统的发育。此外，我们的基因诱捕策略应该适用于斑马鱼的任何发育器官或系统，从而提高斑马鱼作为了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耳或神经特异性基因陷阱线。假型逆转录病毒载体或Tol 2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GAL 4-UAS系统来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来筛选。将创建在机械感觉系统的外周或中枢组分中显示相对特异性表达的细胞系，并克隆捕获的基因。(2)使用基因捕获的Gal 4-驱动系用于体内靶向细胞消融。我们的基因陷阱设计的一个主要优点是它的潜在的靶向生物活性分子在体内特定的细胞，而不需要分离的细胞或组织特异性启动子。这可以通过穿过特定的Gal 4-陷阱线（即，激活子系）与携带置于UAS序列下游的靶基因的转基因系（即，效应器线）。只有当激活物和效应物在同一细胞中都有活性时，效应物蛋白才会表达。Gal 4（GeneSwitch）的可诱导形式将允许对效应蛋白表达的开始进行甚至更多的控制。作为原理证明，将用毒素基因上游的UAS创建效应子系。当与任何驱动系杂交时，我们预计毒素将只特异性杀死那些表达被捕获基因的细胞。这应该被证明是特别强大的选择性消融中枢神经系统神经元的子集，以评估其在发展和/或行为中的作用。我们的新基因陷阱屏幕应促进基因发现斑马鱼，并随时允许测试候选基因参与发展的听觉系统。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。

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.