Cryopreservation and Cloning of Somatic Cells to Preserve Zebrafish Germplasm

体细胞的冷冻保存和克隆以保存斑马鱼种质

• 批准号: 9048319
• 负责人: JOSE B CIBELLI
• 金额: $ 22.5万
• 依托单位: FREEZEBACK, LLC
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9048319
• 关键词: Acceleration; Acute; Adult; Aging; Animal Model; Animals; Back; Backcrossings; Biological Preservation; Biomedical Research; Cardiovascular Diseases; Cells; Cloning; Communities; Cryopreservation; Cryopreserved Cell; Development; Embryo; Embryo Cloning; Emerging Technologies; Female; Fertilization; Fishes; Founder Generation; Freezing; Generations; Genes; Genetic; Germ; Goals; Head; Hematological Disease; Hour; Human; Kidney Diseases; Laboratories; Life; Liquid substance; Malignant Neoplasms; Methods; Modeling; Mus; NIH Program Announcements; Neurodegenerative Disorders; Nitrogen; Nuclear; One-Step dentin bonding system; Organism; Phase; Rattus; Recovery; Research; Research Personnel; Services; Somatic Cell; Source; Sperm Banks; System; Technology; Temperature; Testing; Time; Tissues; Uterus; Work; Zebrafish; cost; genetic makeup; human disease; improved; innovation; male; mutant; new technology; novel; novel strategies; novel therapeutics; offspring; public health relevance; response; sex; somatic cell nuclear transfer; sperm cell; success

 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%.

 描述（由申请人提供）：目前不可能冷冻整个斑马鱼胚胎以恢复活鱼。目前，寻求保存斑马鱼遗传系的研究人员只能选择将冷冻精子储存在精子库中或保持鱼存活。限制增加了成本并阻碍了优秀成果的有效传播 Freeze Back LLC 正在开发一种保存斑马鱼种质的新方法，该方法在受精后 20 至 22 小时内从胚胎中收集体细胞，并将其保存在液氮温度下。通过解冻体细胞并将其用作体细胞核移植（SCNT）的核供体来恢复克隆胚胎发育成雄性和雌性成体，准备开始产生携带原始系的所有基因的后代。两项重要创新的潜力：1) 一种常规、有效保存和恢复斑马鱼种质的通用方法；2) 一步恢复与原始斑马鱼系基因组成完全相同的 F1 代，无需在初步研究中，我们展示了一种新的改进方法，可以从 20 至 22 个 hpf 胚胎中分离出的新鲜体细胞克隆斑马鱼，2- 克隆健康鱼。冷冻保存的细胞，以及从单个冷冻保存的创始人获得的 3 代动物，我们将提供这项新技术作为服务，我们将以有竞争力的成本定期为研究人员储存和恢复任何所需的斑马鱼品种。通过冷冻保存和 SCNT 克隆另外 10 种斑马鱼品系（包括突变体和“弱品系”）来证明这些方法的通用性。我们的短期目标是开发方法（冷冻保存、通过 SCNT 解冻和恢复原始菌株），从而实现斑马鱼的系统存储 我们的长期目标是提供“冷冻返回系统”作为斑马鱼研究界的一项服务，并作为现有冷冻库（例如）的补充。 ZIRC 和 EZRC 将实现以下目标： 目标 I：利用从 AB 和 Tubingen 品系分离的冷冻保存体细胞克隆斑马鱼 里程碑：建立多个第一代创始动物。目标 II：证明通过 SCNT 从冷冻细胞克隆斑马鱼，即创始动物，其后代具有可育性。 里程碑：为两个克隆系产生可育的 F1 后代。目标 III：提高克隆的成功率。里程碑：克隆率达到 5%。