The Francis Crick Institute, Developmental Biology Laboratory, 1 Midland Road, London NW1 1AT, UK.
The Francis Crick Institute, Developmental Biology Laboratory, 1 Midland Road, London NW1 1AT, UK.
Dev Cell. 2018 Mar 12;44(5):597-610.e10. doi: 10.1016/j.devcel.2018.01.022. Epub 2018 Feb 22.
Antisense morpholino oligomers (MOs) have been indispensable tools for developmental biologists to transiently knock down (KD) genes rather than to knock them out (KO). Here we report on the implications of genetic KO versus MO-mediated KD of the mesoderm-specifying Brachyury paralogs in the frog Xenopus tropicalis. While both KO and KD embryos fail to activate the same core gene regulatory network, resulting in virtually identical morphological defects, embryos injected with control or target MOs also show a systemic GC content-dependent immune response and many off-target splicing defects. Optimization of MO dosage and increasing incubation temperatures can mitigate, but not eliminate, these MO side effects, which are consistent with the high affinity measured between MO and off-target sequence in vitro. We conclude that while MOs can be useful to profile loss-of-function phenotypes at a molecular level, careful attention must be paid to their immunogenic and off-target side effects.
反义吗啉代寡核苷酸（MOs）已成为发育生物学家瞬时敲低（KD）基因而非敲除（KO）基因的不可或缺的工具。在这里，我们报告了在青蛙 Xenopus tropicalis 中，中胚层特异性 Brachyury 同源基因的遗传 KO 与 MO 介导的 KD 的影响。尽管 KO 和 KD 胚胎都未能激活相同的核心基因调控网络，导致几乎相同的形态缺陷，但用对照或靶标 MO 注射的胚胎也显示出系统的 GC 含量依赖性免疫反应和许多非靶标剪接缺陷。MO 剂量的优化和孵育温度的升高可以减轻，但不能消除这些 MO 的副作用，这与体外测量到的 MO 和非靶序列之间的高亲和力一致。我们得出结论，虽然 MO 可以在分子水平上有用地研究功能丧失表型，但必须注意它们的免疫原性和非靶标副作用。
