Grants and Contracts Details
Description
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.
Status | Finished |
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Effective start/end date | 8/15/21 → 7/31/24 |
Funding
- Office of the Director: $415,790.00
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