Development of long-term preservation and revival protocols for Drosophila

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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.