Optimization of vitrification methods for Drosophila embryos

项目摘要 果蝇研究实验室通常保持数百至数千种独特的菌株，其中每一种新的 每隔4至6周，必须将一代转移到新鲜食物中。这种做法不仅是劳动和资源 密集的，但有价值的菌株将丢失或获得额外的突变的风险。这一限制将 果蝇冷冻保存方法的发展将克服这一问题。因此，本提案的总体目标 是开发一种简单、可靠的方法来冷冻保存果蝇胚胎。根据文献回顾和我们的 在低温生物学和果蝇生物学的专业知识，我们预计，玻璃化是最佳的方法， 冷冻保存玻璃化是一种“无冰”冷冻保存方法，其中细胞负载高浓度的 冷冻保护剂（CPA，例如二甲基亚砜、丙二醇等）的浓度并迅速冷却 通过玻璃化转变。其结果是形成一种无定形的玻璃态，而不是结晶冰。 大约30年前，有报道称有一种玻璃化冷冻野生型果蝇的方法。然而，该协议是 这是一个非常复杂的过程，导致近70%的胚胎在发育成成年人时丢失。但这 突破性的方法是一个很好的起点，我们在这里，我们将采取全面的 通过结合概念和技术来简化和优化玻璃化程序的方法 工业化学工程学和低温生物学在目标1中，我们将简化胚胎透化 程序，然后通过结合微流体使方案的液体处理步骤自动化。自动化 将减少用户错误并减少劳动力需求。在目标2中，我们将通过以下方式优化玻璃化程序： 系统地评估了一系列注册会计师和注册会计师鸡尾酒解决方案。这些信息将用于 增加CPA的胚内浓度，从而降低冷却时冰结晶的风险 或复温。我们最终将合并两个目标的结果，以验证这种简化和优化的玻璃化 我们的目标是使解冻的胚胎存活率达到≥60%。更好地确保 成功完成这里所描述的工作，我们组建了一个多学科的团队，包括 低温生物学家、工程师和果蝇生物学专家。成功完成这一探索性提案 将导致一个简化和强大的玻璃化冷冻方法的发展，果蝇胚胎， 迅速应用于实验室和资源中心。

PROJECT SUMMARY Drosophila research labs routinely maintain hundreds to thousands of unique strains, where each new generation must be transferred to fresh food every 4 to 6 weeks. Not only is this practice labor and resource intensive, but there is a risk that valuable strains will be lost or acquire additional mutations. This limitation would be overcome by the development of Drosophila cryopreservation methods. Thus, the overall goal of this proposal is to develop a simple, robust method to cryopreserve Drosophila embryos. Based on literature review and our expertise in cryobiology and Drosophila biology, we anticipate that vitrification is the optimal approach to cryopreservation. Vitrification is an `ice-free' method of cryopreservation where cells are loaded with high concentrations of cryoprotective agents (CPAs, e.g. dimethyl sulfoxide, propylene glycol, etc.) and rapidly cooled through the glass transition. The result is formation of an amorphous glassy state as opposed to crystalline ice. Nearly thirty years ago, a vitrification protocol for wild-type Drosophila was reported. However, this protocol is quite complex and results in a loss of nearly 70% of embryos upon development into adults. However, this breakthrough method represents an excellent starting point for us here, where we will take a comprehensive approach to both simplify and optimize the vitrification procedure by incorporating concepts and technologies from industrial chemistry, engineering and cryobiology. In Aim 1, we will simplify the embryo permeabilization procedure and then automate the liquid handling steps of the protocol by incorporating microfluidics. Automation will reduce user-error and decrease labor demands. In Aim 2, we will optimize the vitrification procedure by systematically evaluating a series of CPAs and CPA cocktail solutions. This information will then be used to increase the intraembryonic concentration of CPAs, thereby reducing the risk of ice crystallization upon cooling or rewarming. We will finally merge results from both Aims to validate this simplified and optimized vitrification procedure, where our goal is to achieve ≥60% survival of thawed embryos to adults. To better ensure the successful completion the work described here, we have assembled a multidisciplinary team including cryobiologists, engineers and Drosophila biology experts. The successful completion of this exploratory proposal will result in the development of a simplified and robust vitrification method for Drosophila embryos that can be quickly implemented into laboratories and resource centers.