Quantitative assessment of the knockdown efficiency of morpholino antisense oligonucleotides in zebrafish embryos using a luciferase assay
Quantitative assessment of the knockdown efficiency of morpholino antisense oligonucleotides in zebrafish embryos using a luciferase assay
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DOI：
10.1002/dvg.20361
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发表时间：
2008-01-01
期刊：
影响因子：
1.5
通讯作者：
Kondoh, Hisato
中科院分区：
文献类型：
--
作者：
Kamachi, Yusuke;Okuda, Yuichi;Kondoh, Hisato
尽管吗啉代反义寡核苷酸 (MO) 被广泛用于敲低斑马鱼胚胎中的基因功能，但该方法的效率尚未成功评估，特别是在翻译阻断 MO 的情况下。在我们目前的研究中，我们描述了一种基于荧光素酶测定的系统，该系统可以通过使用包含感兴趣基因的 5'-mRNA 序列和荧光素酶编码序列的融合报告构建体来监测斑马鱼中的 MO 敲低水平。与该报告RNA构建体和靶向感兴趣基因的MO共同注射的斑马鱼胚胎中荧光素酶活性的降低与相应内源蛋白质合成的抑制水平以及敲低表型的出现密切相关。这表明我们的方法的有用性。我们还发现，如果这些基因与所讨论的 25 碱基 MO 之间存在 15 个碱基或更长的连续同源性，则 MO 可以对非预期基因靶标产生相当大的敲低效应。然而，我们的定量评估方法还表明，当采用利用低水平使用的双 MO 的协同效应的策略时，可以实现有效且特异性的击倒。
Despite the broad use of morpholino antisense oligonucleoticles (MO) to knockdown gene function in zebrafish embryos, the efficiency of this method has not been successfully assessed, particularly in the cases of translation-blocking MOs. In our current study, we describe a luciferase assay-based system that can monitor the MO knockdown levels in zebrafish by the use of a fusion reporter construct containing the 5'-mRNA sequence of the gene of interest and the luciferase coding sequence. The decrease in luciferase activity in zebrafish embryos that have been coinjected with this reporter RNA construct and a MO that targets the gene of interest correlated well with the level of inhibition of the corresponding endogenous protein synthesis, and also with the appearance of a knockdown phenotype. This indicates the usefulness of our method. We have also found that MOs can exert considerable knockdown effects upon unintended gene targets if 15 bases or longer of contiguous homology exists between these genes and the 25-base MO in question. Our quantitative assessment method also reveals, however, that an effective and specific knockdown can be achieved when employing a strategy that takes advantage of the synergistic effect of double MOs used at low levels.