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有效的药物开发靶点。许多功能基因组学方法，如微阵列，目前正被应用于这一问题，然而这些方法并不直接识别与疾病过程有关的功能基因。Zonomics公司的研究目标之一是开发一种脊椎动物系统，能够直接识别与人类疾病有关的基因和途径。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的有效疾病模型和分析。斑马鱼是目前唯一适合这一目的的脊椎动物。重要的是，大量证据表明，在斑马鱼等低等脊椎动物系统中，许多重要的分子和生理途径是保守的。Zonomics已经在鱼身上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。 为了有效地重复筛选斑马鱼基因组中的药物靶标，我们建议建立一个包含大约100万个定义的、随机分布的逆转录病毒插入的等量精子文库，从而有效地实现基因组的饱和突变。在该计划的第一阶段，我们建议开发一套用于饱和突变过程的逆转录病毒载体(特异性目标1)。这些病毒将被设计成通过插入干扰和/或转录终止来使基因失活，或改变基因表达。我们将开发和鉴定高滴度病毒种群(特定目标2)，然后在第二阶段使用它来创建饱和诱变精子库，并表征与每个鱼类品系相关的插入位点和突变基因。这个文库资源，我们估计可能用于数百个体外受精，然后可以用于疾病模型的大规模筛选和候选基因测试的药物靶标识别。此外，该图书馆将为Zonomics的内部研究以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.