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However, these protocols failed to be adopted due to the lack of reproducibility and protocol complexity requiring specialized devices (Supplementary Table 1, Supplementary Fig. Indeed, other investigators have sought to develop methods to cryopreserve Drosophila embryos using only wildtype strain (i.e., Oregon R) 8, 9. In comparison, cryopreservation of Drosophila melanogaster provides enormous advantages, including protection against genetic drift to stabilize genotypes and allow expanded genetic and evolutionary studies, decreasing stock maintenance costs, and reducing the risk of stock loss caused by contamination or accidental mixing of precious stocks 7, 8, 9. Currently, the stocks must be manually maintained through frequent and costly transfer of breeding adults to fresh food. Not surprisingly, there are >160,000 unique genotypes held in individual research laboratories and stock centers worldwide and this number is growing 7. The fruit fly ( Drosophila melanogaster), a foundational genetic model organism for biological research in the past century, has driven important discoveries leading to countless biomedical science breakthroughs including six Nobel Prizes 1, 2, 3, 4, 5, 6. Lastly, we find that non-specialists are able to use this protocol to obtain consistent results, demonstrating potential for wide adoption. 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. 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 demonstrate that for most strains, >50% embryos hatch and >25% of the resulting larvae develop into adults after cryopreservation.
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We show that the protocol is broadly applicable, successfully implemented in 25 distinct strains from different sources. We present innovations for embryo permeabilization, cryoprotectant agent loading, and rewarming. Here we report a robust and easily implemented cryopreservation protocol of Drosophila melanogaster embryos.
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The development of a widely adopted cryopreservation method remains a major challenge in Drosophila research.