Generation of a zebrafish insertional mutation library

描述(由申请人提供)：目前，不可能快速和廉价地获得所选基因中的脊椎动物基因敲除，也不可能容易地在脊椎动物中进行全基因组正向遗传筛选。这一第二阶段SBIR应用程序的目标是创建在脊椎动物中执行这些功能基因组学实验所需的研究工具，方法是开发一个包含每个可识别基因的标签插入突变的斑马鱼库。这项拟议的研究将集中于创建、存储和管理斑马鱼精子库，称为Zenemark库，包含逆转录病毒介导的斑马鱼基因组每个LENE中的插入突变。这些插入突变中的每一个都将被表征，存储为多个冷冻精子，并使用侧翼基因组序列、斑马鱼基因位点和哺乳动物基因同源物的识别符输入数据库。一组100万个独立的插入片段平均每1800个碱基对就会产生一次标记基因中断，基本上是每个斑马鱼基因的一个突变。该数据库和来自该文库的单个突变菌株将向商业和学术研究人员开放。正如这一资源将允许基础研究人员更容易地研究脊椎动物生物过程的遗传学一样，它将允许制药研究人员将全基因组正向遗传学应用于药物和药物靶标发现的问题。特别是，ZeneMark文库的创建将使制药研究人员能够筛选突变鱼类的集合，例如具有所有可用药基因突变的鱼类，以1)识别新的药物靶标，2)识别可直接用于高通量体内药物发现的人类疾病的斑马鱼模型，以及3)确定已知药物、毒素或其他化学物质的作用部位(S)。这一前瞻性基因筛查将作为该文库商业化的主要基础，Zonomics已经在寻找企业合作伙伴以及风险资本来资助该计划的第三阶段。

DESCRIPTION (provided by applicant): At the present time it is not possible to rapidly and inexpensively obtain a vertebrate knockout in a gene of choice, or to readily perform whole genome forward genetic screening in a vertebrate. The goal of this Phase II SBIR application is to create the research tools necessary to perform these functional genomics experiments in a vertebrate by developing a zebrafish repository containing a tagged insertional mutation in every identifiable gene. The proposed research will focus on creating, storing, and managing a zebrafish sperm library, termed the Zenemark Library, containing retrovirally-mediated insertional mutations in each lene in the zebrafish genome. Each of these insertional mutations will be characterized, stored as multiple =liquots of frozen sperm, and entered in a databank using identifiers of flanking genomic sequence, zebrafish gene locus, and mammalian gene homologues. A collection of 1,000,000 independent inserts will create, on average, a tagged genetic disruption once every 1800 base pairs, essentially a mutation in every zebrafish gene. The database, and individual mutant strains derived from the library will be available to both commercial and academic researchers. Just as this resource will allow basic researchers to more readily study the genetics of biological processes in vertebrates, it will allow pharmaceutical researchers to apply whole genome forward genetics to the problems of drug and drug target discovery. In particular, the creation of the ZeneMark Library will allow pharmaceutical researchers to screen collections of mutant fish, such as fish with mutations in all druggable genes, to 1 ) identify novel drug targets, 2) identify zebrafish models of human disease that may be used directly for high-throughput in vivo drug discovery, and 3) identify the site(s) of action of known drugs, toxins, or other chemicals. This forward genetic screening will serve as the primary basis for commercialization of the library, and Znomics is already in the process of seeking both corporate partners as well as venture capital to fund Phase III of this program.