Enabling Xenopus oocytes and embryos to perform RNAi

使非洲爪蟾卵母细胞和胚胎能够进行 RNAi

• 批准号: 8339842
• 负责人: Michael D Sheets
• 金额: $ 18.81万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-16 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8339842
• 关键词: 3' Untranslated Regions; Address; Adverse effects; Animal Model; Biogenesis; Biology; Biomedical Research; Cells; Cellular biology; Characteristics; Defect; Development; Dominant-Negative Mutation; Embryo; Ensure; Event; Gene Expression; Genes; Goals; Guide RNA; In Vitro; Indium; Injection of therapeutic agent; Messenger RNA; Methods; MicroRNAs; Oocytes; Organism; Phenocopy; Phenotype; Process; Production; Proteins; RNA Interference; RNA-Binding Proteins; Rana; Readiness; Small Interfering RNA; Source; Staging; Structure; Supplementation; System; Testing; Time; Work; Xenopus; Xenopus laevis; Xenopus oocyte; abstracting; base; genetic analysis; human DICER1 protein; insight; loss of function; nuclease; pre-miRNA; relating to nervous system; research study; source guides; tool; xenopus development

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.

描述（由申请人提供）： 摘要 尽管非洲爪蟾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，其实用性受到限制。其他难以进行基因操作的生物体通常通过使用 RNAi 来研究，即灭活 通过使用小干扰 RNA (siRNA) 靶向这些基因的 mRNA 进行降解，从而对特定基因进行降解。然而，RNAi 在非洲爪蟾卵母细胞或早期胚胎中不起作用，因此无法使用 siRNA 来分析非洲爪蟾发育的早期阶段。因此，非常需要支持这些事件研究的新方法或工具。 最近，我们发现非洲爪蟾卵母细胞和早期胚胎缺乏功能性 Ago2，这是一种含有蛋白质的核酸酶，可通过 siRNA 引导至目标 mRNA，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蟾卵母细胞和早期胚胎中进行 RNAi，通过灭活特定基因产物，有可能使它们能够进行遗传分析。 虽然我们的初步结果表明，可以在向细胞补充外源 Ago2 的情况下进行 RNAi，但仍有几个问题需要解决，以确保该方法的稳健性。我们将确定引入细胞中的 Ago2 mRNA 的最佳量、注射的 Ago2 mRNA 3' UTR 中元素的要求以及随后注射 siRNA 的时间，并且我们将量化在各种条件下积累的 Ago2 的量（目标 1） 。在目标 2 中，我们将确定引入细胞中的 siRNA 的最佳量，并确定引导 RNA 的替代来源（例如 shRNA 和结构基于 pre-miRNA-451 的 RNA）的效用和效率，这些 RNA 的结构基于 pre-miRNA-451（经过加工）直接由 Ago2 提供。最后，（目标 3）由于我们的最终目标是将 RNAi 应用于内源性非洲爪蟾 mRNA 进行功能丧失研究，因此我们将在原理验证实验中应用我们优化的 RNAi 条件，重点关注功能丧失表型为已知。如果我们成功地确定了非洲爪蟾卵母细胞和胚胎中有效 RNAi 的条件，通过外源指导 RNA 和 Ago2 靶向这些特定 mRNA，应该会产生通过更费力和更昂贵的方法预测的表型。 公共卫生相关性： 项目叙述 非洲爪蟾是一种广泛用于生物医学研究的模式生物。我们正在开发新工具，将显着提高这个已经很强大的实验系统的实用性。