Development of a novel method for cryopreservation of Drosophila melanogaster

项目摘要 这项建议旨在开发一种资源，用于保护果蝇。这 昆虫是生物学研究的基础模式生物。经过世纪的研究， 已经产生了携带不同突变等位基因或转基因构建体的果蝇品系。不过有一 用果蝇进行研究的局限性在于目前还没有一种实用的方法来冷冻保存果蝇 菌株玻璃化冷冻果蝇的传统方法是在20世纪90年代早期开发的， 由于执行协议的困难而被广泛采用。这是一个从实践角度看的问题 由于所有这些菌株都需要花费大量的成本和劳动力单独地维持在连续培养中， 从科学的角度来看，由于在连续培养的过程中， 并且可能发生污染，降低这些资源对于未来实验的价值。一种新的方法 为果蝇的冷冻保存提出了这个R24资源中心。分离的胚胎细胞核， 将被冷冻保存，然后通过显微注射进行核移植， 从冷冻保存的细胞核中产生克隆。这种方法避免了与 阻止常规冷冻保存的胚胎膜的不渗透性 已经被用于其他生物的方法。胚胎细胞核将使用 自然启发的方法。多样的生物系统（植物、昆虫等）在脱水干旱中存活 冷冻温度和其他压力通过使用渗透剂。在应用层面上，建议 研究有可能通过以下方式改变果蝇品系的保存：1）保存亚细胞 与胚胎相反的组件（特别是细胞核）;以及2）自动化大部分工作流程。在漫长的- 这个资源中心的目标是开发一个强大的和可扩展的低温保存协议， 果蝇，从而降低了成本，提高了长期菌种维持的质量。

PROJECT SUMMARY This proposal seeks to develop a resource for the preservation of the fruit fly, Drosophila melanogaster. This insect is a foundational model organism for biological research. Over a century of work, an enormous number of fly strains harboring different mutant alleles or transgenic constructs have been generated. However, one limitation of working with flies is that there is as yet no practical method for cryopreservation of Drosophila strains. Conventional methods of vitrifying Drosophila were developed in the early 1990s and were never widely adopted due to the difficulty in performing the protocols. This is a problem from a practical perspective since all these strains need to be individually maintained in continuous culture at substantial cost and labor, and also from a scientific perspective, since in the process of continuous culture mutations can accumulate and contamination can occur, degrading the value of these resources for future experiments. A novel approach for cryopreservation of Drosophila is proposed for this R24 resource center. Isolated embryonic nuclei, rather than intact embryos, will be cryopreserved and then nuclear transplantation via microinjection will be used to create clones derived from the cryopreserved nuclei. This approach avoids the issues associated with the impermeability of embryonic membranes that have prevented the use of conventional cryopreservation approaches that have been used with other organisms. Embryonic nuclei will be cryopreserved using a naturally inspired approach. Diverse biological systems (plants, insects, etc.) survive dehydration, drought, freezing temperatures and other stresses through the use of osmolytes. On an applied level, the proposed investigation has the potential to transform preservation of Drosophila lines by 1) preserving subcellular components (specifically nuclei) as opposed to embryos; and 2) automating much of the workflow. In the long- term, the goal of this resource center is to develop a robust and scalable protocol for cryopreservation of Drosophila, thus reducing the cost and improving the quality of long-term strain maintenance.