Development of a novel method for cryopreservation of Drosophila melanogaster

项目总结 这项提议寻求开发一种资源来保存果蝇，黑腹果蝇。这 昆虫是生物学研究的基础模式生物。一个多世纪的工作，一个巨大的数字 已经产生了携带不同突变等位基因或转基因构建物的苍蝇品系。然而，有一个 利用果蝇进行研究的局限性在于，目前还没有实用的果蝇超低温保存方法 菌株。传统的玻璃化冷冻果蝇的方法是在20世纪90年代初开发出来的，从未 由于执行协议的困难而被广泛采用。从实际的角度来看，这是一个问题。 由于所有这些菌株都需要以相当高的成本和劳动力在连续培养中单独保持， 而且从科学的角度来看，因为在不断的培养过程中，突变可以积累 而且可能会发生污染，降低这些资源在未来实验中的价值。一种新的方法 对R24资源中心果蝇的超低温保存提出了建议。分离的胚胎细胞核，而不是 将被冷冻保存，然后通过显微注射的核移植将被用于 创建从冷冻保存的细胞核衍生的克隆。这种方法避免了与 阻止使用常规冷冻保存的胚胎膜的不透性 已经被用于其他生物体的方法。胚胎细胞核将通过一种 自然启发的方法。多样的生物系统(植物、昆虫等)在脱水、干旱中幸存下来， 冰冻温度和通过使用渗透分子造成的其他压力。在应用层面上，拟议的 研究有可能通过1)保存亚细胞来改变果蝇品系的保存 组件(特别是细胞核)，而不是胚胎；以及2)使大部分工作流程自动化。从长远来看- 该资源中心的目标是开发一种健壮且可扩展的冷冻保存协议 果蝇，从而降低了成本，提高了长期品系维护的质量。

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.