Usefulness of multiple chalk-based food colorings for inducing better gene silencing by feeding RNA interference in planarians
Usefulness of multiple chalk-based food colorings for inducing better gene silencing by feeding RNA interference in planarians
复制标题
多种粉笔基食用色素通过对涡虫进行 RNA 干扰来诱导更好的基因沉默
DOI：
10.1111/dgd.12413
复制
发表时间：
2018
影响因子：
2.5
通讯作者：
Umesono Y.
中科院分区：
文献类型：
--
作者：
Hattori;M.;Miyamoto;M.;Hosoda;K.;Umesono Y.
涡虫已被广泛认为是无脊椎动物再生研究的主要动物模型之一。为了通过进食诱导涡虫的RNA干扰（RNAi），广泛接受的方案是让动物喂食两到三次含有双链RNA （dsRNA）和可见食用色素（如血液）的食物，以确认个体动物的进食。然而，一个可能的问题是，添加的食用色素通常在肠道内保留数天，这使得很难根据喂食前后肠道内颜色密度的差异来确认每轮dsRNA喂养的成功。因此，在接受dsRNA喂养的个体之间，食欲水平的差异导致它们之间由于敲除不足而产生表型变异。在我们试图克服这个问题的过程中，我们开发了一种新的方法，通过将三种不同颜色的粉笔（粉色、黄色和蓝色）作为食用色素的组合，在多次喂食的个体中实现对dsRNA喂养成功的强有力确认。值得注意的是，我们发现这种方法优于传统方法，可以对那些在每天一次喂食的四次中主动食用含有dsRNA的食物的个体进行积极标记。使用这些选择的动物，我们获得了稳定和足够强的RNAi诱导表型。我们将这种改良的多色白垩针喂食RNAi的方法命名为“Candi”，并提出其在涡虫基因功能分析中的益处。
Planarians have become widely recognized as one of the major animal models for regeneration studies in invertebrates. To induce RNA interference (RNAi) by feeding in planarians, the widely accepted protocol is one in which animals undergo two or three feedings of food containing double‐stranded RNA (dsRNA) plus visible food coloring (e.g., blood) for confirmation of feeding by individual animals. However, one possible problem is that incorporated food coloring is often retained within the gut for several days, which makes it difficult to confirm the success of each round of dsRNA feeding based on the difference of the color density within the gut before and after feeding. As a consequence, the difference of appetite levels among individuals undergoing dsRNA feeding leads to phenotypic variability among them due to insufficient knockdown. In our attempts to overcome this problem, we have developed a novel method for achieving robust confirmation of the success of dsRNA feeding in individuals fed multiple times by means of including a combination of three different colored chalks (pink, yellow and blue) as food coloring. Notably, we found that this method is superior to the conventional method for positively marking individuals that actively consumed the dsRNA‐containing food during four times of once‐daily feeding. Using these selected animals, we obtained stable and sufficiently strong RNAi‐induced phenotypes. We termed this improved multi‐colored chalk‐spiked method of feeding RNAi “Candi” and propose its benefits for gene function analysis in planarians.