Egg Cooling After Oviposition Extends the Permissive Period for Microinjection-Mediated Genome Modification in Bombyx mori

In general, transgenesis efficiency is largely dependent on the developmental status of eggs for microinjection. We investigated whether the relationship between transgenesis efficiency and cooling eggs in silkworms, <i>Bombyx mori</i>, affects the transgenesis frequencies. First, we performed a microinjection using eggs of different developmental statuses at 25 °C. As a result, the use of eggs at 4 h after egg-laying (hAEL) demonstrated nearly five times greater efficiency in frequency compared to 8 hAEL but no transgenesis was found at 12 hAEL. Second, we examined the use of eggs stored for 5 or 24 h at 10 °C. We found that transgenic silkworms were produced not only 5 hAEL but also 24 hAEL. Finally, in the <i>BmBLOS2</i> gene knock-out experiment, eggs stored at 10 °C demonstrated knock-out phenotypes even 48 hAEL at the time of injection (G₀). These results demonstrate that an egg cooling treatment enables drastically enhanced rates of efficiency for insect genome modification. Our results could be useful in other insects, especially species with an extremely short syncytial preblastodermal stage.