Development of Germplasm Resources for Preservation of Aquatic Models

描述（由申请人提供）：在过去的十年中，世界各地的实验室已经生产了数以万计的突变、转基因和野生型鱼种。维持这些有价值的基因型是昂贵的，有风险的，甚至超过了最大的库存中心的能力。2007年NCRR研讨会“实现高通量生物医学种质保存库”的主要建议之一是进行详细研究，以改善重要水族鱼模型的精子冷冻保存。在此基础上，我们建议通过扩展全面的冷冻保存途径，提高效率和产量，为种质资源库的实施建立高通量处理斑马鱼精子的库存中心和实验室。具体目标是：1)拓宽途径，以提高质量，并为现有方法生产的样品提供多个接入点；2)加强自动化处理和存储库开发的途径，包括使用工业建模和决策工具；3)实施两种可扩展的低温保存协议；资源中心使用的大规模高通量方法，并从它进行逆向工程，为其他研究实验室提供小型到中等规模的应用程序，以备份他们自己的生产线或将它们提交给资源中心。这将提高质量，减少可变性，并提高效率。我们建议改进亲鱼调节、配子质量、受精和繁殖，以适应各种方法、应用水平以及研究界当前和未来的需求。这将产生两种互补的方法：一种是在工业规模上使用自动化设备和汇集样本，以产生最高水平的强度和效率；另一种，不那么密集但高度可靠，设计或传播到其他实验室。从这条外部通道产生的材料将进入中央通道，并将根据来自多个地点的总产出提供额外形式的吞吐量。高水平的标准化对于两种方法有效地合并，将高质量的样品交付到大型综合种质资源库是必要的。因此，我们将这一更新设想为一个漏斗，以改进和适应适用于斑马鱼冷冻保存的广泛技术方法，并将这些活动集中在存储库开发上，最终可以与商业上可用的高通量设备、用品和专门针对观赏鱼优化的试剂相结合。这种方法是实际可行的。这个更新建议直接建立在我们已建立的途径的成功基础上，并期望与未来的进步有效地结合起来。

DESCRIPTION (provided by applicant): In the past decade, laboratories around the world have produced tens of thousands of mutant, transgenic, and wild-type fish lines. Maintaining these valuable genotypes is expensive, risky, and beyond the capacity of even the largest stock centers. One of the major recommendations of the 2007 NCRR workshop 'Achieving High-Throughput Repositories for Biomedical Germplasm Preservation' was for performance of detailed studies to improve sperm cryopreservation for important aquarium fish models. Pursuant to this we propose to establish high-throughput processing of zebrafish sperm for stock centers and laboratories by extending a comprehensive cryopreservation pathway, and by intensifying efficiency and output to enable implementation of germplasm repositories. The Specific Aims are to: 1) broaden the pathway to improve quality and provide multiple access points for samples produced by current approaches, 2) intensify the pathway for automated processing and repository development including use of industrial modeling and decision-making tools, and 3) implement two scalable cryopreservation protocols; the large-scale high throughput method for Resource Center use and, reverse-engineered from it, a small to intermediate scale application for other research laboratories to back up their own lines or submit them to the Resource Center. This will improve quality, reduce variability, and increase efficiency. We propose to improve broodstock conditioning, gamete quality, fertilization and reproduction, and to accommodate the variety of approaches, levels of application, and current and future needs of the research community. This will yield two complementary approaches: one at an industrial scale using automated equipment and pooling of samples to yield the highest level of intensity and efficiency; the other, less intensive but highly reliable, designed or dissemination to other laboratories. The materials generated from this external pathway will feed into the central pathway and will provide an additional form of throughput based on the aggregate production from multiple locations. A high level of standardization will be necessary for both approaches to merge efficiently to deliver high-quality samples into large comprehensive germplasm repositories. Thus, we envision this renewal as a funnel to improve and adapt the wide breadth of technical approaches available for zebrafish cryopreservation, and to focus these activities for repository development that can be eventually coupled with commercially available high-throughput equipment, supplies, and reagents specifically optimized for aquarium fish. This approach is practical and extremely feasible. This renewal proposal builds directly on the success of our established pathway and looks to couple efficiently with future advances.