Cryopreservation method for Drosophila melanogaster embryos

Li Zhan,Min-Gang Li,John Bischof,Thomas Hays

doi:10.1038/s41467-021-22694-z

Li Zhan, Min-Gang Li + Show 2 more

Open Access

https://doi.org/10.1038/s41467-021-22694-z

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Journal: Nature Communications	Publication Date: Apr 23, 2021
Citations: 24	License type: open-access

Affiliation: University of Minnesota

Abstract

The development of a widely adopted cryopreservation method remains a major challenge in Drosophila research. Here we report a robust and easily implemented cryopreservation protocol of Drosophila melanogaster embryos. We present innovations for embryo permeabilization, cryoprotectant agent loading, and rewarming. We show that the protocol is broadly applicable, successfully implemented in 25 distinct strains from different sources. We demonstrate that for most strains, >50% embryos hatch and >25% of the resulting larvae develop into adults after cryopreservation. We determine that survival can be significantly improved by outcrossing to mitigate the effect of genetic background for strains with low survival after cryopreservation. We show that flies retain normal sex ratio, fertility, and original mutation after successive cryopreservation of 5 generations and 6-month storage in liquid nitrogen. Lastly, we find that non-specialists are able to use this protocol to obtain consistent results, demonstrating potential for wide adoption.

Highlights

The development of a widely adopted cryopreservation method remains a major challenge in Drosophila research
We established that 22 h old embryos provided the highest post cryopreservation survival, which corresponds to early stage 16 when head involution and dorsal closure have been completed (Fig. 2a)[20]
As post cryopreservation survival depends on embryo age upon vitrification, strains with limited egg retention could potentially have higher post cryopreservation survival rates

Summary

Introduction

The development of a widely adopted cryopreservation method remains a major challenge in Drosophila research. Other investigators have sought to develop methods to cryopreserve Drosophila embryos using only wildtype strain (i.e., Oregon R)[8,9] These protocols failed to be adopted due to the lack of reproducibility and protocol complexity requiring specialized devices (Supplementary Table 1, Supplementary Fig. 1); no protocol has been successfully implemented to cryopreserve a broad collection of Drosophila stocks, both wildtype and mutant[7]. The major challenges to cryopreserve Drosophila melanogaster embryos include cryoprotectant agent (CPA) loading, vitrification with scalability, embryo age-dependent survival, and straindependent genetic backgrounds. To tackle the above mentioned challenges, we introduce innovations especially focusing on permeabilization, CPA loading and vitrification, leading to a simple and robust cryopreservation protocol supporting wide adoption by the Drosophila community (Supplementary Table 1)

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