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万种突变、转基因和野生型鱼类品系。保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容量储存库”的主要建议之一是进行详细的研究，以改善重要水族馆鱼类模型的精子冷冻保存。根据这一点，我们建议建立高通量处理的斑马鱼精子的股票中心和实验室，通过扩展一个全面的冷冻保存途径，并通过提高效率和输出，使实施种质资源库。具体目标是：1）拓宽途径以提高质量并为通过当前方法产生的样品提供多个接入点，2）强化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，以及3）实施两种可扩展的低温保存方案;用于资源中心的大规模高吞吐量方法，并从其反向工程，一个小到中等规模的应用程序，供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。这将提高质量，减少变化，并提高效率。我们建议改善亲鱼调理，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：一种是工业规模的方法，使用自动化设备和样本池，以产生最高水平的强度和效率;另一种是强度较低但高度可靠的方法，设计或传播到其他实验室。从该外部路径产生的材料将进料到中央路径中，并且将基于来自多个位置的聚集体生产提供额外形式的吞吐量。高水平的标准化将是必要的，这两种方法有效地合并，提供高质量的样品到大型综合种质库。因此，我们设想这种更新作为一个漏斗，以改善和适应广泛的技术方法，可用于斑马鱼冷冻保存，并集中这些活动的存储库开发，最终可以与市售的高通量设备，用品和试剂，特别是优化水族馆鱼类。这种做法是切实可行的。这一更新建议直接建立在我们既定途径的成功基础上，并期待与未来的进步有效结合。

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.