Development of Germplasm Resources for Preservation of Aquatic Models

描述（由申请人提供）：迫切需要改善对水生模式生物（如斑马鱼和medaka）遗传资源的保护。通过冷冻保存获得的研究品系将深刻地影响毒理学、胚胎学、遗传学、药物开发和人类疾病等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种突变的、转基因的和野生型的鱼种。然而，这些线路的实时维护是昂贵的，有风险的，并且超出了即使是最大的库存中心目前的能力。尽管冷冻保存是一种长期保存遗传物质的有效方法，但目前的鱼类冷冻保存方案尚未标准化，结果不一致，并威胁到大规模遗传筛选的有效性。为了使精子冷冻保存成为一种可靠、经济的鱼类基因库工具，需要改进整个冷冻保存过程，并将其整合到一个高效的大规模平台中，该平台将遗传和生物数据库、档案存储能力、库存管理和样本分发途径联系起来。我们的项目目标是通过优化、简化和标准化程序，并应用基本的冷冻生物学和生理学参数，建立高通量的冷冻保存斑马鱼和medaka精子，以供储存中心和研究实验室使用。我们将评估以下因素的影响：1)动物调节，2)样品收集，3)精子浓度，4)扩展剂组成，5)添加剂，6)冷藏和运输，7)冷冻保护剂毒性，8)冷却速率和冷冻保护剂的相互作用，9)解冻，10)解冻后修正，11)受精程序。我们将建立高通量样品管理：1)吸管填充和密封，2)标签，3)库存管理，4)运输可靠性，5)采用高安全性塑料吸管的自动化处理。我们将为库存中心和捐赠实验室的质量控制和生物安全定义术语并使程序标准化。种质低温保存将通过以下方式极大地促进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体动物的效率；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.