199 Oral - Techniques and Technology Session
Saturday April 09, 5:45 PM - 6:00 PM

Authors:
Li Zhan; Min-gang Li; Amanda Neisch; Thomas Hays; John Bischof

Affiliation: University of Minnesota, Minneapolis, MN

Keywords:
q. other (cryopreservation); r. other (embryo cryopreservation)

The fruit fly (Drosophila melanogaster), a foundational genetic model organism in the past century, has driven important biomedical science breakthroughs including six Nobel Prizes. However, more than 160,000 unique genotypes worldwide must be manually maintained through frequent and costly transfer of breeding adults to fresh food. In comparison, cryopreservation provides enormous advantages including protection against genetic drift, decreasing stock maintenance costs, and reducing the risk of stock loss caused by contamination or accidental mixing of precious stocks. Here we report an easily implemented and robust cryopreservation protocol of Drosophila melanogaster embryos, demonstrating potential for wide adoption. We present innovations for embryo permeabilization, cryoprotectant agent (i.e., anti-freezer) loading, and rewarming. After embryo collection, our protocol takes ~35 min to freeze and store the embryos in liquid nitrogen, ~25 min to thaw the embryo and remove the intra-embryonic cryoprotectant agent. We showcase its implementation in 25 distinct strains including wildtype and mutants. Using this protocol, >50% embryos hatch and >25% of the resulting larvae develop into adults (i.e., survival normalized to untreated embryos) after cryopreservation for most strains. For strains with initially low survival after cryopreservation, we show that the survival can be substantially improved by outcrossing to mitigate the effect of genetic background. After successive cryopreservation of 5 generations and 12-month storage in liquid nitrogen, the flies retain normal sex ratio, fertility and original mutation. Importantly, we find that non-specialists are able to use this protocol to obtain consistent results after training.