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）： 虽然非洲爪蟾是一种功能强大的模式生物，为脊椎动物的发育、细胞生物学和神经生物学提供了重要的机制性见解，但由于缺乏遗传分析和基因操作，它的实用性受到限制。其他难以遗传操纵的生物体通常通过使用RNA干扰（使基因失活）进行研究。 通过使用靶向这些基因的mRNA进行降解的小干扰RNA（siRNA），然而，RNAi在非洲爪蟾卵母细胞或早期胚胎中不起作用，使得不可能在非洲爪蟾发育的早期阶段分析中使用siRNA。因此，非常需要新的方法或工具来支持对这些事件的研究。 最近，我们发现非洲爪蟾卵母细胞和早期胚胎缺乏功能性Ago 2，这是一种含有核酸酶的蛋白质，可以通过siRNA引导靶向mRNA，这可以解释为什么这些细胞无法进行RNAi。幸运的是，我们还发现由注射的体外合成mRNA产生的外源性Ago 2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，通过灭活特定的基因产物，使它们能够进行遗传分析。 虽然我们的初步结果表明，RNAi可以在用外源性Ago 2补充细胞后进行，但仍有几个问题有待解决，以确保该方法是稳健的。我们将确定待引入细胞的Ago 2 mRNA的最佳量、对注射的Ago 2 mRNA的3' UTR中的元件的要求以及随后注射siRNA的时机，并且我们将定量在各种条件下积累的Ago 2的量（目标1）。在目标2中，我们将确定引入细胞的siRNA的最佳量，并确定指导RNA的替代来源如shRNA和其结构基于前体miRNA-451的RNA的效用和效率，其直接由Ago 2加工。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的功能丧失研究，我们将在原理验证实验中应用我们优化的RNAi条件，重点关注其功能丧失表型已知的内源性mRNA。如果我们已经成功地确定了在非洲爪蟾卵母细胞和胚胎中进行有效RNAi的条件，那么通过外源性指导RNA和Ago 2靶向这些特异性mRNA应该会产生通过更费力和昂贵的方法预测的表型。 公共卫生关系： 项目简介非洲爪蟾是一种广泛用于生物医学研究的模式生物。我们正在开发新的工具，这将显着增加这个已经强大的实验系统的效用。