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（由适用提供）：目前无法恢复整个斑马鱼胚胎以恢复活鱼。寻求保护斑马鱼遗传线的研究人员仅限于在精子库中存储冷冻精子或维持鱼类的选择。这些限制增加了成本，并阻碍了上级的有效传播 研究人员之间的野生型和突变菌株。 Freeze Back LLC正在开发一种新的方法来保存斑马鱼种植质量。受精后20至22小时从胚胎收集体细胞，并在液氮温度下储存。通过解冻体细胞并将它们用作核细胞核转移（SCNT）来回收活鱼。克隆的胚胎生长成雄性和女性成年人，准备开始产生原始线的所有基因的后代。这项技术具有两项重要创新的潜力1）一种常规和有效保存和恢复斑马鱼种质 - 和2）F1一代的一般方法，其基因组成与原始斑马鱼的遗传构成完全相同，而无需回到创始人动物的情况下。在初步研究中； 1-证明了一种从20至22 hpf胚胎分离的新鲜体细胞中克隆斑马鱼的新方法，从冷冻保存细胞中分离出2-2种克隆的健康鱼，以及从单个冷冻保存创始人获得的两种男女的3个产生的动物。我们将提供这项新技术作为一项服务。我们将以有竞争力的成本常规存储并为研究人员恢复任何所需的斑马鱼菌株。在第二阶段，我们将通过冷冻保存和克隆的另外10种斑马鱼菌株（包括突变体和“弱菌株”）来证明这些方法的通用性。我们的短期目标是开发能够系统地融入斑马鱼的方法（冷冻保存，解冻和恢复原始菌株）。我们的长期目标是将“冻结系统”作为斑马鱼研究界的服务，并作为当前现有的Cryobanks的补充，例如ZIRC和EZRC。我们将执行以下目标：目标I：从AB和Tubingen菌株中分离出的冷冻保存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。 AIM II：证明斑马鱼是由冷冻细胞（即创始人动物）和后代肥沃的斑马鱼克隆的。里程碑：为两个克隆线产生肥沃的F1后代。 AIM III：提高克隆的成功率。里程碑：达到5％的克隆率。