Generation of a zebrafish insertional mutation library

描述（由申请人提供）：目前，不可能快速、低成本地获得脊椎动物基因敲除，也不可能在脊椎动物中容易地进行全基因组正向遗传筛选。该二期SBIR申请的目标是创建必要的研究工具，通过开发斑马鱼储存库，在每个可识别的基因中包含标记的插入突变，从而在脊椎动物中进行这些功能基因组学实验。拟议的研究将侧重于创建、存储和管理一个称为Zenemark库的斑马鱼精子库，该库包含斑马鱼基因组中每个lene中逆转录病毒介导的插入突变。每一个插入突变都将被描述，储存为多个冷冻精子，并使用侧面基因组序列、斑马鱼基因位点和哺乳动物基因同源物的标识符输入数据库。100万个独立插入的集合平均每1800个碱基对就会产生一个被标记的基因中断，基本上在每个斑马鱼基因中都有一个突变。该数据库以及从该文库中获得的单个突变株将对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的集合，例如所有可药物基因突变的鱼，以1)鉴定新的药物靶点，2)鉴定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)鉴定已知药物，毒素或其他化学物质的作用部位。这种前瞻性基因筛选将成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资本，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.