Simple and Cost-effective Storage of Mouse Sperm using Evaporative Drying

使用蒸发干燥法简单且经济高效地储存小鼠精子

• 批准号: 8740910
• 负责人: JOHN D BIGGERS
• 金额: $ 68.68万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-16 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8740910
• 关键词: Animal Model; Animals; Biological; Biological Preservation; Breeding; Cell membrane; Cells; Cryopreservation; Desiccation; Development; Disease; Embryo; Embryonic Development; Engineering; Equipment; Ethics; Exposure to; Freeze Drying; Freezing; Future; Genetic; Genotype; Germ Cells; Goals; Health; Influentials; Knock-out; Laboratories; Life; Living Costs; Logistics; Maintenance; Mammalian Cell; Methodology; Methods; Modeling; Molecular Genetics; Motivation; Mus; Mutant Strains Mice; Oocytes; Organism; Policies; Process; Production; Protocols documentation; Recovery; Reporting; Reproductive Technology; Research; Research Personnel; Shipping; Ships; Study models; Techniques; Temperature; Training; Transgenic Organisms; Translating; Trehalose; Work; animal care; cold temperature; cost; cost effective; cryogenics; design; egg; human disease; improved; in vivo; mouse genome; mutant; mutant mouse model; offspring; public health relevance; repository; sperm cell

DESCRIPTION (provided by applicant): The rapid advances in molecular genetics and reproductive technology during the past decade have led to the production of a large number of artificially produced animal genotypes. The synergy of these two fields has been especially influential in the mouse, since it is now the primary animal model for many human diseases. It is important, therefore, to reliably preserve the large numbers of mouse genotypes for use by future investigators as safely, simply, and economically as possible. Traditionally, mouse genotypes are stored by maintaining active breeding colonies of mutant mice. However, the cost of live colony maintenance has become prohibitively expensive. Alternatively, the banking of spermatozoa would be an efficient and cost effective approach for the storage of transgenic and mutant stocks. Several methods are available today for cryopreservation and maintenance of mouse sperm, but these require sophisticated freezing equipment and methodologies. More recently, freeze-drying for storage at ambient temperatures have been developed to circumvent some of the limitations of cryogenic storage. However, freeze-drying also relies on sophisticated and cumbersome equipment. Thus, there is still a pressing need to develop stable repositories of mouse sperm stored at ambient temperatures with simplicity for seamless dissemination. Evaporative drying offers a simpler, faster, and inexpensive approach that can be mastered quickly and accomplished successfully with minimal training. Furthermore, cellular stability is achieved quickly at ambient temperature without prolonged exposure to deleterious conditions during drying. Organisms that naturally enter a dried state during dormancy do so by evaporative drying, which suggests evaporative drying may be innocuous to cells under proper conditions. In Specific Aim 1, we will optimize both the physicochemical and biological conditions to achieve high recovery of sperm after desiccation and long-term storage at ambient temperature. In Specific Aim 2, we will develop engineered trehalose-derivatives that freely permeates the plasma membrane of the sperm for efficient loading of the protectant at amounts necessary for stabilization. In Specific Aim 3, we will analyze the viability, health, and genetic and phenotypic fidelity of embryos and live born offspring generated from evaporatively dried sperm stored at ambient temperatures.

描述（由申请人提供）：在过去的十年中，分子遗传学和生殖技术的快速进步导致了大量人工产生的动物基因型的产生。这两个领域的协同作用在小鼠中特别有影响力，因为它现在是许多人类疾病的主要动物模型。因此，重要的是要可靠地保留大量的小鼠基因型，以便在未来的研究人员中尽可能安全，简单，经济地使用。传统上，小鼠基因型是通过维持突变小鼠的活跃繁殖菌落来存储的。但是，实时殖民地维护的成本变得非常昂贵。另外，精子的储备将是一种有效且具有成本效益的方法，用于存储转基因和突变股。如今，有几种方法可用于冷冻保存和维护小鼠精子，但这些方法需要精致的冰冻设备和方法。最近，已经开发了在环境温度下存储的冷冻干燥，以规避低温存储的某些局限性。但是，冷冻干燥也依赖于复杂且繁琐的设备。因此，仍然需要迫切需要开发稳定的小鼠精子存储在环境温度下的小鼠精子，以简单地进行无缝传播。蒸发干燥提供了一种更简单，更快，廉价的方法，可以通过最少的培训来快速掌握并成功完成。此外，在环境温度下迅速实现了细胞稳定性，而在干燥过程中却长时间暴露于有害条件下。在休眠期间，自然进入干燥状态的生物是通过蒸发干燥的，这表明在适当条件下细胞可能无害。在特定目标1中，我们将优化物理化学和生物学条件，以在周围温度下进行干燥和长期储存后获得高恢复精子。在特定的目标2中，我们将开发工程的树形衍生物，这些衍生物可以自由渗透到精子的质膜，以有效地以稳定的量为其有效加载保护剂。在特定的目标3中，我们将分析胚胎的生存力，健康以及遗传和表型保真度以及由蒸发干燥的精子产生的，该后代存储在环境温度下。