Development of Germplasm Resources for Preservation of Aquatic Models

说明(申请人提供)：迫切需要加强对斑马鱼和青竹等水生模式生物遗传资源的保存。通过低温保存的研究路线的可用性将深刻影响毒理学、胚胎学、遗传学、药物开发和人类疾病方面的生物医学研究的进展。在过去的15年里，世界各地的实验室已经生产了数千条突变、转基因和野生类型的鱼线。然而，这些线路的现场维护既昂贵又有风险，甚至超过了最大的库存中心目前的能力。虽然冷冻保存是一种被证明是长期保存遗传物质的方法，但目前的FISH方案并不标准化，结果不一致，并威胁到大规模遗传筛查的有效性。为了使精子冷冻保存成为鱼类遗传库的可靠、成本效益高的工具，需要改进整个冷冻保存过程，并将其整合到一个有效的大规模平台中，该平台与遗传和生物数据库、档案存储能力、库存管理和样品分发路径相连接。我们的项目目标是通过优化、简化和标准化程序以及应用基本的低温生物学和生理参数，建立用于库存中心应用和研究实验室使用的斑马鱼和青竹精子的高通量冷冻保存。我们将评估以下影响：1)动物条件，2)样本采集，3)精子密度，4)延长剂成分，5)添加剂，6)冷藏储存和运输，7)冷冻保护剂毒性，8)降温速度和冷冻保护剂的相互作用，9)解冻，10)解冻后修改，11)受精程序。我们将建立高通量样品管理：1)秸秆灌装和封口，2)标签，3)库存管理，4)运输可靠性，5)高安全性塑料吸管的自动化加工。我们将为库存中心和捐赠者实验室的质量控制和生物安全定义术语和标准化程序。种质超低温保存将极大地加强NIH资源的开发、管理和分配：1)减少设施的规模和生产成本，2)保护遗传多样性和减少近亲繁殖，3)最大限度地提高饲养活动物的效率，3)促进遗传物质的全球、区域和机构运输。

DESCRIPTION (provided by applicant): There is an immediate need to improve preservation of genetic resources from aquatic model organisms such as zebrafish and medaka. The availability of research lines through cryopreservation will profoundly influence the advancement of biomedical research in toxicology, embryology, genetics, drug development, and human disease. In the past 15 years, laboratories around the world have produced thousands of mutant, transgenic and wild-type fish lines. However, live maintenance of these lines is expensive, risky, and beyond the current capacity of even the largest stock centers. Although cryopreservation is a proven method for long-term maintenance of genetic material, current protocols for fish are not standardized, yield inconsistent results, and threaten the efficacy of large-scale genetic screening. For sperm cryopreservation to become a reliable, cost-effective tool for genetic banking of fish, the overall cryopreservation process needs to be improved and integrated into an efficient large-scale platform that links with genetic and biological databases, archival storage capabilities, inventory management, and sample distribution pathways. Our Project Goal is to establish high-throughput cryopreservation of zebrafish and medaka sperm for stock center application and for research laboratory use by optimizing, streamlining, and standardizing procedures, and by applying basic cryobiological and physiological parameters. We will evaluate the effects of: 1) animal conditioning, 2) sample collection, 3) sperm concentration, 4) extender composition, 5) additives, 6) refrigerated storage and transport, 7) cryoprotectant toxicity, 8) interactions of cooling rate and cryoprotectants, 9) thawing, 10) post-thaw amendments, and 11) fertilization procedures. We will establish high-throughput sample management of: 1) straw-filling and sealing, 2) labeling, 3) inventory management, 4) shipping reliability, and 5) adoption of automated processing of high-security plastic straws. We will define terms and standardize procedures for quality control and biosecurity at stock centers and donor laboratories. Germplasm cryopreservation will greatly enhance development, management, and distribution of NIH resources by: 1) reducing size and production costs of facilities, 2) protecting genetic diversity and reducing inbreeding, 3) maximizing efficiency of holding live animals, and 3) facilitating global, regional, and institutional transport of genetic material.