Development of long-term preservation and revival protocols for Drosophila

项目总结：该项目将改进模式生物的超低温保存方法 果蝇，从而减少了维持多样性所需的时间和资源 基因股票。果蝇成功冷冻保存的两个主要挑战是 通过胚胎膜促进低温保护剂的摄取和克服毒性 这些低温保护性化合物。为了应对这些挑战，我们将双管齐下 接近。首先，我们将使用饲料添加剂来增加果蝇胚胎的冷冻保护剂。第二, 我们将优化使用声旋转法进一步增强低温保护分子的方法。 并为胚胎加载晚期胚胎发生丰富蛋白(S)(LEAPS)，这是一类 防止渗透压力和促进耐冻性的蛋白质。优化后 装载防冻剂和LEAP，我们将不同的冻结和解冻速度来识别 能够成功地将活苍蝇从超低温中恢复的条件。 具体目标和研究设计：具体目标1：提高 将冷冻保护剂引入胚胎的声波手术。我们设计了一种改良的食谱 这会增加胚胎中的海藻糖和脯氨酸，这里将优化声旋转法以 进一步提高冷冻防护剂水平。Sonopation结合使用超声波频率 具有脂质微泡，在细胞中产生小孔，并推动溶质的吸收。我们将使用 荧光分子来优化声波修饰方法，而这些优化后的方法将 用于添加冷冻保护剂和将LEAP加载到胚胎中。Leap之前已经 已被证明能提高黑腹果蝇细胞系的应激耐受性。 具体目标2：研究冷冻和解冻策略，以改善冷冻保存。 我们将测试携带冷冻保护剂的苍蝇胚胎在各种冷冻/解冻中存活的能力 费率。将探索三种不同的冷冻(随后解冻)苍蝇胚胎的策略。 利用已建立的昆虫超低温保存方法。 意义：该项目将显著提高冷冻防护的效率 分子可以被引入黑腹水母的胚胎中。当前的冷冻保存方法 在技术上具有挑战性，大多数实验室都无法接触到。这里开发的方法将 为克服胚胎对许多人的不渗透性提供常规、廉价的策略 冷冻保护剂。成功的策略将在多种常用基因中进行测试 背景和突变菌株，以测试这些保存策略在多大程度上 对不同的基因类型有效。

Project Summary: This project will improve methods for cryopreserving the model organism Drosophila melanogaster, thereby reducing the time and resources needed to maintain diverse genetic stocks. Two of the primary challenges for successful cryopreservation in Drosophila are promoting uptake of cryoprotectants through embryonic membranes and overcoming the toxicity of these cryoprotective compounds. To address these challenges, we will use a two-pronged approach. First, we will use dietary additives to increase cryoprotectants in fly embryos. Second, we will optimize methods for using sonoporation to further augment cryoprotective molecules and load embryos with LATE EMBRYOGENESIS ABUNDANT PROTEIN(s) (LEAPs), a class of proteins that protect against osmotic stress and promote freeze tolerance. After optimizing loading of cryoprotectants and LEAPs, we will vary freezing and thawing rates to identify conditions that can successfully recover live flies from ultralow temperatures. Specific aims and research design: Specific Aim 1: Improving the efficacy of sonoporation to introduce cryoprotectants into embryos. We have devised a modified diet that increases trehalose and proline in embryos, and here will optimize sonoporation methods to further augment cryoprotectant levels. Sonoporation uses ultrasonic frequencies in conjunction with lipid microbubbles to generate small pores in cells and drive uptake of solutes. We will use fluorescent molecules to optimize sonoporation methods, and these optimized methods will then be used to load augment cryoprotectants and load LEAPs into embryos. LEAPs have previously been shown to increase stress tolerance of D. melanogaster cell lines. Specific Aim 2: Investigate freezing and thawing strategies to improve cryopreservation. We will test the ability of cryoprotectant-loaded fly embryos to survive various freezing/thawing rates. Three different strategies to freeze (and subsequently thaw) fly embryos will be explored using established methods for insect cryopreservation. Significance: This project will significantly improve the efficiency by which cryoprotective molecules can be introduced into D. melanogaster embryos. Current cryopreservation methods are technically challenging and inaccessible to most labs. The methods developed here will provide routine, inexpensive strategies for overcoming the impermeability of embryos to many cryoprotectants. Successful strategies will be tested in multiple commonly used genetic backgrounds and mutant strains, to test the extent to which these preservation strategies are effective in different genotypes.