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脊椎动物所选基因的敲除，或容易地在脊椎动物中进行全基因组正向遗传筛选。这个第二阶段SBIR应用的目标是通过开发一个在每个可识别基因中含有标记插入突变的斑马鱼库，创建在脊椎动物中进行这些功能基因组学实验所需的研究工具。拟议的研究将集中在创建，存储和管理斑马鱼精子库，称为Zenemark库，包含斑马鱼基因组中每个lene的逆转录病毒介导的插入突变。将表征这些插入突变中的每一个，将其储存为冷冻精子的多个=液量，并使用侧翼基因组序列、斑马鱼基因座和哺乳动物基因同源物的标识符输入数据库。平均而言，100万个独立插入物的集合将每1800个碱基对产生一次标记的遗传破坏，基本上是每个斑马鱼基因的突变。商业和学术研究人员都可以使用该数据库以及从该库中获得的个别突变菌株。正如该资源将使基础研究人员能够更容易地研究脊椎动物生物过程的遗传学一样，它将使制药研究人员能够将全基因组正向遗传学应用于药物和药物靶点发现的问题。特别是，ZeneMark文库的创建将允许制药研究人员筛选突变鱼类的集合，例如所有可药用基因突变的鱼类，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斑马鱼模型，以及3）识别已知药物，毒素或其他化学品的作用位点。这种正向基因筛选将作为该文库商业化的主要基础，Znomics已经在寻求企业合作伙伴和风险投资，为该计划的第三阶段提供资金。

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.