Liu Wei-Yi, Wang Yan, Sun Yong-Hua, Wang Yun, Wang Ya-Ping, Chen Shang-Ping, Zhu Zuo-Yan
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China.
Dev Growth Differ. 2005 Jun;47(5):323-31. doi: 10.1111/j.1440-169X.2005.00807.x.
Double-stranded RNA (dsRNA) has been shown to be a useful tool for silencing genes in zebrafish (Danio rerio), while the blocking specificity of dsRNA is still of major concern for application. It was reported that siRNA (small interfering RNA) prepared by endoribonuclease digestion (esiRNA) could efficiently silence endogenous gene expression in mammalian embryos. To test whether esiRNA could work in zebrafish, we utilized Escherichia coli RNaseIII to digest dsRNA of zebrafish no tail (ntl), a mesoderm determinant in zebrafish and found that esi-ntl could lead to developmental defects, however, the effective dose was so close to the toxic dose that esi-ntl often led to non-specific developmental defects. Consequently, we utilized SP6 RNA polymerase to produce si-ntl, siRNA designed against ntl, by in vitro transcription. By injecting in vitro synthesized si-ntl into zebrafish zygotes, we obtained specific phenocopies of reported mutants of ntl. We achieved up to a 59%no tail phenotype when the injection concentration was as high as 4 microg/microL. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization analysis showed that si-ntl could largely and specifically reduce mRNA levels of the ntl gene. As a result, our data indicate that esiRNA is unable to cause specific developmental defects in zebrafish, while siRNA should be an alternative for downregulation of specific gene expression in zebrafish in cases where RNAi techniques are applied to zebrafish reverse genetics.
双链RNA（dsRNA）已被证明是一种在斑马鱼（Danio rerio）中使基因沉默的有用工具，而dsRNA的阻断特异性仍是应用中主要关注的问题。据报道，通过核糖核酸内切酶消化制备的小干扰RNA（siRNA，esiRNA）可有效沉默哺乳动物胚胎中的内源基因表达。为了测试esiRNA在斑马鱼中是否有效，我们利用大肠杆菌RNaseIII消化斑马鱼中胚层决定因子无尾（ntl）的dsRNA，发现esi-ntl可导致发育缺陷，然而，有效剂量与毒性剂量非常接近，以至于esi-ntl常常导致非特异性发育缺陷。因此，我们利用SP6 RNA聚合酶通过体外转录产生针对ntl设计的si-ntl（siRNA）。通过将体外合成的si-ntl注射到斑马鱼受精卵中，我们获得了已报道的ntl突变体的特异性拟表型。当注射浓度高达4μg/μL时，我们获得了高达59%的无尾表型。定量逆转录-聚合酶链反应（RT-PCR）和全胚胎原位杂交分析表明，si-ntl可大量且特异性地降低ntl基因的mRNA水平。因此，我们的数据表明，esiRNA在斑马鱼中无法引起特异性发育缺陷，而在将RNAi技术应用于斑马鱼反向遗传学的情况下，siRNA应该是下调斑马鱼中特定基因表达的一种替代方法。
