Too much interference: Injection of double-stranded RNA has nonspecific effects in the zebrafish embryo
Too much interference: Injection of double-stranded RNA has nonspecific effects in the zebrafish embryo
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DOI：
10.1006/dbio.2000.9761
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发表时间：
2000-08-01
影响因子：
2.7
通讯作者：
Ho, RK
中科院分区：
文献类型：
--
作者：
Oates, AC;Bruce, AEE;Ho, RK
我们研究了双链RNA（dsRNA）抑制脊椎动物斑马鱼（Danio rerio）基因表达的能力。将对应于T-box基因tbx/16/spadetail（spt）的dsRNA注射到早期野生型胚胎中，导致囊胚中tbx 16/spt mRNA的快速和戏剧性的损失。依赖tbx 16/spt功能表达的papc、tbx 6和gata 1基因的mRNA减少，显然是模仿了spt突变表型。然而，来自许多不受spt突变影响的基因的mRNA，如β连环蛋白、stat 3和无尾，也丢失了，这表明tbx 16/spt dsRNA的“干扰”效应不限于内源性tbx 16/spt mRNA。我们比较了注射来自斑马鱼tbx 16/spadetail、nieuwkoid/bozozok和Brachyury/no tail基因的dsRNA与来自细菌lacZ基因的dsRNA的效果。在每一种情况下，胚胎显示在受精后12和24小时的异常的变量综合征。在盲法研究中，我们无法区分各种dsRNA的作用。与dsRNA的共同作用一致，无论序列如何，注射来自lacZ基因的dsRNA同样有效地强烈降低囊胚中的tbx 16/spt和β连环蛋白mRNA。这些研究结果表明，尽管已发表的报告，目前的双链RNA干扰的方法是不是一个实用的技术，在早期斑马鱼发育合子基因功能的调查。(C)北京大学出版社.
We have investigated the ability of double-stranded RNA (dsRNA) to inhibit gene expression in a vertebrate, the zebrafish, Danio rerio. Injection of dsRNA corresponding to the T-box gene tbx/16/spadetail (spt) into early wild-type embryos caused a rapid and dramatic loss of tbx16/spt mRNA in the blastula. mRNAs from the papc, tbx6, and gata1 genes, which depend on tbx16/spt function for their expression, were reduced, apparently mimicking the spt mutant phenotype. However, mRNAs from a number of genes that are unaffected by the spt mutation, such as beta catenin, stat3, and no tail, were also lost, indicating that the "interference" effect of tbx16/spt dsRNA was not restricted to the endogenous tbx16/spt mRNA. We compared the effects of injecting dsRNA from the zebrafish tbx16/spadetail, nieuwkoid/bozozok, and Brachyury/no tail genes with dsRNA from the bacterial lacZ gene. In each case the embryos displayed a variable syndrome of abnormalities at 12 and 24 h postfertilization. In blind studies, we could not distinguish between the effects of the various dsRNAs. Consistent with a common effect of dsRNA, regardless of sequence, injection of dsRNA from the lacZ gene was likewise effective in strongly reducing tbx16/spt and beta catenin mRNA in the blastula. These findings indicate that, despite published reports, the current methodology of double-stranded RNA interference is not a practical technique for investigating zygotic gene function during early zebrafish development. (C) 2000 Academic Press.