Generation of a zebrafish insertional mutation Library

描述（由申请人提供）：许多重要的人类疾病缺乏药物开发的有效靶点。许多功能基因组学的方法，如微阵列，目前正在应用于这个问题，但这些方法并不直接识别基因的功能参与疾病的过程。Znomics，Inc.的研究目标之一是，是开发一种能够直接识别与人类疾病有关的基因和途径的脊椎动物系统。为了实现这一目标并确定新的药物靶点，我们计划将全基因组正向遗传学应用于斑马鱼（Danio rerio）的经验证的疾病模型和测定。斑马鱼是目前唯一适合于这一目的的脊椎动物。重要的是，大量的证据表明，许多重要的分子和生理途径在低等脊椎动物系统，如斑马鱼的保护。Znomics已经在鱼类中获得了两个经过验证的人类疾病模型，目前正在创建另外六个模型。 为了以有效的方式重复筛选斑马鱼基因组中的药物靶点，我们建议创建一个等分的精子文库，其中包含约1，000，000个定义的、随机分布的逆转录病毒插入片段，以便有效地实现基因组的饱和诱变。在该计划的第一阶段，我们建议开发一套用于饱和诱变程序的逆转录病毒载体（特定目标1）。这些病毒将被设计为通过插入破坏和/或转录终止来复制基因，或改变基因表达。我们将开发和表征高滴度病毒储备液（特定目标2），然后将其用于第2阶段，以创建饱和诱变精子文库，并表征插入位点，从而表征与每种鱼类品系相关的突变基因。我们估计这个库资源可以用于数百次体外受精，然后可以用于疾病模型中的大规模筛选以及候选基因测试中的药物靶点鉴定。此外，该图书馆还将为Znomics内部研究以及涉及其他公司和学术研究人员的合作研究提供资源。

DESCRIPTION (provided by applicant): Validated targets for drug development are lacking for many important human diseases. Many functional genomics methods, such as microarray, are currently being applied to this problem, however these methods do not directly identify genes that are functionally involved in disease processes. One goal of the research at Znomics, Inc. is to develop a vertebrate system with the capacity to directly identify genes and pathways involved in human disease. To accomplish this goal and identify new drug targets, we plan to apply whole genome forward genetics to validated disease models and assays in the zebrafish, Danio rerio. The zebrafish is currently the only vertebrate organism well suited to this purpose. Importantly, substantial evidence indicates conservation of many important molecules and physiological pathways across lower vertebrate systems such as the zebrafish. Znomics has already obtained two validated human disease models in the fish, and is currently in the process of creating six additional models. In order to repetitively screen the zebrafish genome for drug targets in an efficient way, we propose to create an aliquoted sperm library containing approximately 1,000,000 defined, randomly distributed, retroviral insertions, so as to effectively achieve saturation mutagenesis of the genome. In the first phase of this program, we propose to develop a set of retroviral vectors (Specific Aim 1) for use in the saturation mutagenesis procedure. These viruses will be designed to inactivate genes by insertional disruption and/or transcriptional termination, or to alter gene expression. We will develop and characterize high titer virus stock (Specific Aim 2) which will then be used in phase 2 to create the saturation mutagenesis sperm library, and characterize the insertion sites, and hence mutated genes, associated with each fish strain. This library resource, which we estimate may be used for several hundred in vitro fertilizations, can then be used for drug target identification in both large scale screens in disease models as well as in candidate gene testing. Furthermore, the library will provide a resource for both internal Znomics research as well as for collaborative research involving other companies as well as academic investigators.