Cryopreservation and Cloning of Somatic Cells to Preserve Zebrafish Germplasm

 描述(申请人提供)：冷冻整个斑马鱼胚胎以便以后恢复活鱼目前是不可能的。寻求保存斑马鱼基因系的研究人员目前仅限于将冷冻精子存储在精子库中或保持斑马鱼活着的选择。这些限制增加了成本并阻碍了SUBERVER的有效传播 调查人员中的野生型和突变型菌株。冻回有限责任公司正在开发一种保存斑马鱼种质的新方法。在受精后20至22小时从胚胎中收集体细胞，并将其储存在液氮温度下。活鱼是通过解冻体细胞并将其用作体细胞核移植(SCNT)的核供体来回收的。克隆胚胎发育成雄性和雌性成体，准备开始产生携带原始品系所有基因的后代。这项技术具有两项重要创新的潜力：1)斑马鱼种质的常规和有效保存和回收的通用方法；2)与原始斑马鱼品系完全相同的基因组成的F1代，一步就能恢复，而不需要与创始人动物回交。在初步研究中，我们有：1-展示了一种新的和改进的方法来克隆斑马鱼，从20到22个HPF胚胎分离的新鲜体细胞中克隆斑马鱼，2-从冷冻保存的细胞中克隆健康的鱼，以及从单个冷冻保存的创建者获得的3种性别的动物。我们将以服务的形式提供这项新技术。我们将以具有竞争力的成本为研究人员存储和回收任何所需的斑马鱼品系。在第二阶段，我们将通过对另外10个斑马鱼品系(包括突变品系和弱品系)的超低温保存和SCNT克隆来展示这些方法的一般性。我们的短期目标是开发方法(用SCNT冷冻保存、解冻和恢复原始品系)，使斑马鱼能够系统地储存。我们的长期目标是为斑马鱼研究社区提供“冷冻系统”作为服务，并作为现有冷藏库的补充，例如Zirc和EZRC。主要完成以下工作：目的一：从AB和Tubingen品系分离的体细胞中克隆斑马鱼。里程碑：为这两条线建立多个第一代创始人动物。目的II：证明SCNT从冷冻细胞克隆的斑马鱼及其后代是可育的。里程碑：为两个克隆系产生可育的F1后代。目的三：提高克隆成功率。里程碑：实现5%的克隆率。

 DESCRIPTION (provided by applicant): Freezing whole zebrafish embryos in order to later recover live fish is not currently possible. Researchers seeking to preserve zebrafish genetic lines are presently limited to the options of storing frozen sperm in sperm banks or maintaining the fish alive. These limitations increase costs and hinder the efficient dissemination of superior wild type- and mutant strains among investigators. Freeze Back LLC is developing a new approach to the preservation of zebra fish germ-plasm. Somatic cells are collected from embryos at 20 to 22 hours post fertilization and stored at liquid nitrogen temperatures. Live fish are recovered by thawing the somatic cells and using them as nuclear donors for somatic cell nuclear transfer (SCNT). Cloned embryos grow into male and female adults, ready to start producing offspring that carry all the genes of the original line. This technology holds the potential for two important innovations 1) a general method for the routine and effective preservation and recovery of zebra fish germ-plasm and 2) an F1 generation with exactly the same genetic makeup as the original zebrafish line recovered in one step without the need for backcrossing the founder animals. In preliminary studies we have; 1- demonstrated a new and improved method to clone zebrafish from fresh somatic cells isolated from 20 to 22 hpf embryos, 2- cloned healthy fish from cryopreserved cells, and 3- generated animals of both sexes obtained from a single cryopreserved founder. We will offer this new technology as a service. We will routinely store and recover any desired zebrafish strain for researchers at a competitive cost. In Phase II we will demonstrate the generality of these methods by cryopreservation and SCNT cloning of an additional 10 zebra fish strains (including mutant and "weak strains"). Our short-term goal is to develop methods (cryopreservation, thawing and recovery of the original strain by SCNT) that enable the systematic banking of zebra fish. Our long-term goal is to deliver the "Freeze Back system" as a service to the zebrafish research community and as a supplement to currently existing cryobanks e.g ZIRC and EZRC. We will carry out the following aims: Aim I: Clone zebrafish from cryopreserved somatic cells isolated from AB and Tubingen strains. Milestone: Establish multiple first-generation founder animals for these two lines. Aim II: Demonstrate that zebrafish cloned by SCNT from frozen cells, i.e. founder animals, and their offspring are fertile. Milestone: Generate fertile F1 progeny for the two cloned lines. Aim III: Improve the success rate of cloning. Milestone: Achieve a cloning rate of 5%.