Enabling Xenopus oocytes and embryos to perform RNAi

描述(由申请人提供)： 摘要非洲爪哇是一种强大的模式生物，为脊椎动物发育、细胞生物学和神经生物学提供了重要的机制见解，但由于缺乏遗传分析和基因操作，其应用受到限制。其他难以通过基因操作的生物体经常通过使用RNAi进行研究，RNAi是指 通过使用针对特定基因的mRNAs的小干扰RNA(SiRNAs)进行降解。然而，RNAi在非洲爪哇的卵母细胞或早期胚胎中不起作用，因此不可能使用siRNAs来分析非洲爪哇发育的早期阶段。因此，迫切需要新的方法或工具来支持对这些事件的研究。最近，我们发现非洲爪哇卵母细胞和早期胚胎缺乏功能性的Ago2，Ago2是一种含有核酸酶的蛋白，由siRNAs引导到靶mRNAs，这可以解释为什么这些细胞无法进行RNAi。幸运的是，我们还发现通过注射体外合成的mRNA产生的外源Ago2克服了这一缺陷。因此，我们现在可以在非洲爪哇卵母细胞和早期胚胎中进行RNAi，通过使特定基因产物失活来潜在地使它们易于进行遗传分析。虽然我们的初步结果表明，RNAi可以在补充外源Ago2的细胞时进行，但仍有几个问题需要解决，以确保该方法的健壮性。我们将确定将Ago2mRNA导入细胞的最佳数量、对注入Ago2mRNA的3‘UTR区中元件的要求以及随后注射siRNA的时机，并将量化不同条件下Ago2的积累量(目标1)。在目标2中，我们将确定要引入细胞的最佳siRNA量，并确定替代来源的引导RNA的效用和效率，例如shRNA，以及其结构基于前-miRNA-451的RNA，这些RNA由Ago2直接处理。最后，(目标3)由于我们的最终目标是将RNAi应用于内源非洲爪哇mRNAs的功能丧失研究，我们将在原则性实验中应用我们优化的RNAi条件，重点研究其功能丧失表型已知的内源mRNAs。如果我们已经成功地确定了在非洲爪哇卵母细胞和胚胎中进行有效RNAi的条件，那么通过外源引导RNAs和Ago2靶向这些特定的mRNAs应该会产生用更费力和昂贵的方法预测的表型。 公共卫生相关性： 项目简介非洲爪蛙是一种被广泛用于生物医学研究的模式生物。我们正在开发新的工具，这些工具将显著增加这个已经很强大的实验系统的效用。

DESCRIPTION (provided by applicant): Abstract Although Xenopus laevis is a powerful model organism that has provided critical mechanistic insights into vertebrate development, cell biology and neural biology its utility has been limited by a lack of genetic analysis and gene manipulation. Other organisms that are difficult to manipulate genetically have often been studied by the use of RNAi, the inactivation of specific genes through the use of small interfering RNAs (siRNAs) that target the mRNAs of these genes for degradation. However, RNAi does not work in Xenopus oocytes or early embryos, making it impossible to use siRNAs in the analysis of early stages of Xenopus development. Thus new methods or tools that would support the study of these events are greatly needed. Recently, we discovered that Xenopus oocytes and early embryos lack functional Ago2, the nuclease containing protein that is guided to targeted mRNAs by siRNAs, which would explain why these cells are unable to carry out RNAi. Fortunately, we have also found that exogenous Ago2 generated from injected in vitro synthesized mRNA overcomes this deficiency. Thus we can now perform RNAi in Xenopus oocytes and early embryos, potentially making them amenable to genetic analysis through inactivation of specific gene products. While our preliminary results show that RNAi can be performed upon supplementation of cells with exogenous Ago2, several issues remain to be addressed, to ensure that the method is robust. We will determine optimal amounts of Ago2 mRNA to be introduced into the cells, requirements for elements in the 3' UTR of injected Ago2 mRNA and timing of subsequent injection of siRNA and we will quantify the amounts of Ago2 accumulating under various conditions (Aim 1). In Aim 2 we will determine optimal amounts of siRNA to be introduced into the cells and determine the utility and efficiency of alternative sources of guide RNAs such as shRNAs, and RNAs whose structures are based on that of pre-miRNA-451, which are processed by Ago2 directly. Finally, (Aim 3) as our ultimate goal is to apply RNAi to endogenous Xenopus mRNAs for loss of function studies, we will apply our optimized conditions for RNAi in proof-of-principle experiments, focusing on endogenous mRNAs whose loss of function phenotypes are known. If we have successfully identified conditions for efficient RNAi in Xenopus oocytes and embryos, targeting these specific mRNAs by exogenous guide RNAs and Ago2 should produce phenotypes predicted by the more laborious and expensive methods. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE The frog Xenopus laevis is a model organism that is used extensively for biomedical research. We are developing new tools that will significantly increase the utility o this already powerful experimental system.