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.
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.