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Xenopus laevis是一种强大的模型生物体，它为脊椎动物发育，细胞生物学和神经生物学提供了关键的机械洞察力，其效用受到缺乏遗传分析和基因操纵的限制。其他难以操纵基因的生物经常通过使用RNAi来研究 通过使用小型干扰RNA（siRNA）来靶向这些基因降解的特定基因。但是，RNAi在爪蟾卵母细胞或早期胚胎中不起作用，因此无法在Xenopus发育的早期分析中使用siRNA。因此，非常需要支持这些事件研究的新方法或工具。 最近，我们发现爪蟾卵母细胞和早期胚胎缺乏功能性AGO2，含有蛋白质的核酸酶，该蛋白质由siRNA引导至靶向mRNA，这可以解释为什么这些细胞无法执行RNAi。幸运的是，我们还发现，由注射的体外合成mRNA产生的外源AGO2克服了这种缺陷。因此，我们现在可以在爪蟾卵母细胞和早期胚胎中执行RNAi，从而有可能通过灭活特定基因产物来适应遗传分析。 虽然我们的初步结果表明，可以在补充外源AGO2的细胞后进行RNAi，但仍有几个问题待解决，以确保该方法稳健。我们将确定要引入细胞中的AGO2 mRNA的最佳量，对注射AGO2 mRNA的3'UTR中元素的要求以及随后注入siRNA的时间，我们将量化在各种条件下积累的AGO2的量（AIM 1）。在AIM 2中，我们将确定要引入细胞中的siRNA的最佳量，并确定指南RNA的替代源的效用和效率，例如SHRNA（SHRNA）和其结构基于MIRNA-451的RNA，这些结构基于MIRNA-451，这些结构是由Ago 2直接处理的。最后，（AIM 3）作为我们的最终目标是将RNAi应用于内源性爪蟾mRNA，以损失功能研究，我们将在原则证明实验中应用优化的RNAi条件，重点是内源性mRNA，其功能表型的丧失是已知的。如果我们成功地鉴定了异武卵母细胞和胚胎中有效RNAi的条件，则通过外源指南RNA和AGO2靶向这些特定的mRNA，应产生更费力和昂贵的方法预测的表型。 公共卫生相关性： 项目叙事青蛙爪蟾laevis是一种模型生物体，可广泛用于生物医学研究。我们正在开发新工具，这些工具将大大提高这个已经有力的实验系统的实用性。