Development of long-term preservation and revival protocols for Drosophila

果蝇长期保存和复兴方案的制定

基本信息

批准号：
10352620
负责人：
NICHOLAS TEETS
金额：
$ 23.16万
依托单位：
UNIVERSITY OF KENTUCKY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-15 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10352620
关键词：
Address Animal Model Animals Arthropods Biological Cell Line Cells Collaborations Communities Cost Savings Cryopreservation Data Desiccation Development Devices Diet Disease Drosophila genus Drosophila melanogaster Embryo Embryonic Development Engineering Erythrocytes Freeze Drying Freezing Frequencies Genes Genetic Genetic Research Genetic Transformation Genotype Goals Homo sapiens Human Insecta Interdisciplinary Study Lipids Measures Membrane Methods Microbubbles Microfluidics Molecular Molecular Weight Mutation Nature Organism Postdoctoral Fellow Preparation Process Production Proline Proteins Protocols documentation Research Research Design Resources Seeds Stress Technology Temperature Testing Time Toxic effect Trehalose Ultrasonics Water authority cold temperature cost dietary dietary manipulation dietary supplements experience fly improved mutant novel strategies nucleic acid delivery preservation prevent solute sonoporation stress tolerance success training opportunity undergraduate student uptake

项目摘要

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.

项目概要：该项目将改进冷冻保存模式生物的方法果蝇，从而减少维持多样性所需的时间和资源遗传库存。果蝇成功冷冻保存的两个主要挑战是促进胚胎膜吸收冷冻保护剂并克服毒性这些冷冻保护化合物。为了应对这些挑战，我们将采用两管齐下的方法方法。首先，我们将使用膳食添加剂来增加果蝇胚胎中的冷冻保护剂。第二，我们将优化使用声孔技术进一步增强冷冻保护分子的方法并用晚期胚胎发生丰富的蛋白质（LEAP）（一类防止渗透压并促进冷冻耐受性的蛋白质。优化后冷冻保护剂和 LEAP 的加载，我们将改变冷冻和解冻速率来确定可以从超低温中成功回收活蝇的条件。具体目标和研究设计：具体目标1：提高疗效超声穿孔将冷冻保护剂引入胚胎中。我们设计了一种改良饮食增加胚胎中的海藻糖和脯氨酸，这里将优化声孔方法进一步提高冷冻保护剂水平。声孔结合使用超声波频率与脂质微泡一起在细胞中产生小孔并促进溶质的吸收。我们将使用荧光分子来优化声孔方法，然后这些优化的方法将用于加载增强冷冻保护剂并将 LEAP 加载到胚胎中。 LEAP 此前曾已被证明可以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。我们将测试加载冷冻保护剂的果蝇胚胎在各种冷冻/解冻中存活的能力费率。将探索三种不同的冷冻（随后解冻）果蝇胚胎的策略使用昆虫冷冻保存的既定方法。意义：该项目将显着提高冷冻保护的效率分子可以被引入黑腹果蝇胚胎中。目前的冷冻保存方法在技术上具有挑战性并且大多数实验室无法实现。这里开发的方法将提供常规、廉价的策略来克服胚胎对许多人的不渗透性冷冻保护剂。成功的策略将在多种常用遗传基因中进行测试背景和突变菌株，以测试这些保存策略的程度对不同基因型均有效。