Uchino Keiro, Waizumi Ryusei, Sumitani Megumi, Sakai Hiroki, Yamada Nobuto, Kojima Katsura, Yonemura Naoyuki, Tatematsu Ken-Ichiro, Iizuka Tetsuya, Sezutsu Hideki, Tamura Toshiki
Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, 1-2 Owashi, Tsukuba 305-8634, Ibaraki, Japan.
Independent Researcher, Tsukuba 300-1207, Ibaraki, Japan.
Int J Mol Sci. 2024 Nov 25;25(23):12642. doi: 10.3390/ijms252312642.
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, , 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 gene knock-out experiment, eggs stored at 10 °C demonstrated knock-out phenotypes even 48 hAEL at the time of injection (G). 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.
一般来说，转基因效率在很大程度上取决于用于显微注射的卵的发育状态。我们研究了家蚕中转基因效率与卵冷却之间的关系是否会影响转基因频率。首先，我们在25℃下使用不同发育状态的卵进行显微注射。结果表明，产卵后4小时（hAEL）的卵的使用频率效率比8 hAEL的卵高出近五倍，但在12 hAEL时未发现转基因现象。其次，我们检查了在10℃下储存5或24小时的卵的使用情况。我们发现，不仅在5 hAEL时产生了转基因家蚕，在24 hAEL时也产生了。最后，在基因敲除实验中，在10℃下储存的卵在注射时即使在48 hAEL时也表现出敲除表型（G）。这些结果表明，卵冷却处理能够显著提高昆虫基因组修饰的效率。我们的结果可能对其他昆虫有用，特别是那些具有极短合胞体前胚盘阶段的物种。
