Development of Germplasm Resources for Preservation of Aquatic Models

描述(申请人提供)：在过去的十年里，世界各地的实验室已经生产了数万条突变、转基因和野生类型的鱼线。维持这些有价值的基因型是昂贵的、有风险的，甚至超出了最大的库存中心的能力。2007年NCRR研讨会“实现生物医学种质保存的高通量储存库”的主要建议之一是开展详细研究，以改进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超低温保存途径，并通过提高效率和产量来实现种质资源库的实施，为种群中心和实验室建立斑马鱼精子的高通量加工。其具体目标是：1)拓宽提高质量的途径，并为当前方法产生的样本提供多个访问点；2)加强自动化处理和储存库开发的途径，包括使用工业建模和决策工具；3)实施两个可扩展的冷冻保存协议；大规模高通量方法用于资源中心，并在此基础上进行逆向工程，为其他研究实验室备份自己的生产线或将其提交给资源中心提供中小型应用程序。这将提高质量，减少变异性，提高效率。我们建议改善亲鱼条件、配子质量、受精和繁殖，并适应各种方法、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并汇集样本，以产生最高水平的强度和效率；另一种是不那么密集但高度可靠的、设计或传播到其他实验室的方法。这一外部途径产生的物质将进入中央途径，并将基于来自多个地点的总产量提供另一种形式的产量。要使这两种方法有效地合并，将高质量的样本送入大型综合种质储存库，就必须有较高水平的标准化。因此，我们将这次更新设想为一个漏斗，以改进和适应斑马鱼冷冻保存可用的广泛技术方法，并将这些活动集中于储存库的开发，这些储存库最终可以与商业上可用的高通量设备、供应品和专门针对水族鱼优化的试剂相结合。这种方法是实用的，也是非常可行的。这项更新建议直接建立在我们既定途径的成功基础上，并期待与未来的进展有效结合。

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.