Small RNA interactions with transgenes in genetically modified mosquito lines

项目概要： 使用转基因蚊子控制病媒传播疾病的策略依赖于 可以编码新表型的合成转基因的活性，如灭蚊或病毒阻断 表型观察到转基因可能意外地失去功能，这与转基因的调控是一致的。 通过一种称为皮尔纳途径的RNA干扰机制来干扰外源DNA。小RNA 在具有这种功能丧失的埃及伊蚊和斯氏按蚊的转基因系中进行测序 表型揭示了siRNA和piRNA对转基因的不同程度的靶向。这个目标 这项研究是为了鉴定蚊子体内潜在的小RNA对转基因的活性，并直接测试蚊子体内的小RNA是否 这种小RNA可以抑制转基因活性。第一个目的是通过富集来测定siRNA和piRNA。 以及蚊子阶段和解剖组织中与转基因活性相关的小RNA测序 在两个方面，aegypti和An. Stephensi野生型和转基因品系，并整合数据 沿着已发表的转基因序列一起纳入生物信息学资源， 其他实验室和环境。这将是一个主要的产出资源，可以让研究人员设计 避免小RNA识别。第二个目的是直接测试siRNA和piRNA是否 在转基因品系中检测到的是抑制性的;我们将在每个物种中使用转基因传感器菌株进行研究 背景，Ae. aegypti和An.史蒂芬西该菌株将包含一个带有荧光标记的基因组拷贝 被设计成可以被特定的小RNA检测到。如果小RNA是抑制性的， 传感器菌株和测试菌株将导致荧光降低。如果siRNA和piRNA负责 对于这种表型，siRNA和皮尔纳生物发生蛋白DICER 2或小西葫芦的敲低分别将 拯救荧光蛋白表达。来自该交叉的活性也将通过测试以下物质的丰度来测定： 报告基因转录本以及通过测序针对报告基因产生的小RNA，其可以提供 关于阻遏是在转录水平还是转录后水平的信息。总的来说，这项研究将 开始确定小RNA靶向在现有转基因品系中的广度和影响，并奠定 基金会，以探讨如何在野生蚊子的RNA干扰将作出反应，实施转基因 控制蚊媒疾病的技术。

Project Summary: Strategies to use genetically modified mosquitoes for applications to control vector-borne disease rely on the activity of a synthetic transgene that can encode new phenotypes, such as mosquito-killing or virus blocking phenotypes. Observations that transgenes can unexpectedly lose functionality is consistent with regulation of the foreign DNA by an RNA interference mechanism called the piRNA pathway. Preliminary small RNA sequencing in transgenic line of both Aedes aegypti and Anopheles stephensi with such loss of function phenotypes has revealed targeting of transgenes by siRNAs and piRNAs to different degrees. The goal of this study is to identify potential small RNA activity against transgenes in mosquitoes and to directly test whether such small RNA can repress transgene activity. The first aim is to assay for siRNAs and piRNAs by enrichment and small RNA sequencing in the mosquito stages and dissected tissues that are relevant to transgene activity and inheritance in both Ae. aegypti and An. stephensi wild-type and transgenic lines and to integrate that data along with published transgene sequences into a bioinformatics resource that will support hypothesis testing in other labs and contexts. A major output of this will be a resource that can allow researchers to design constructs to avoid small RNA recognition. The second aim is to directly test whether siRNAs and piRNAs detected in transgenic lines are repressive; we will do this using a transgenic sensor strain in each species background, Ae. aegypti and An. stephensi. The strain will contain a genomic copy of with a fluorescent marker designed to be detected by specific small RNAs. If small RNAs are repressive, the offspring of a cross between the sensor strain and the test strain will result in decreased fluorescence. If siRNA and piRNAs are responsible for this phenotype, knockdown of siRNA and piRNA biogenesis proteins DICER2 or Zucchini respectively will rescue fluorescent protein expression. Activity from this cross will also be assayed by testing the abundance of reporter transcripts as well as small RNAs produced against the reporter by sequencing, which can provide information on whether repression is at the transcriptional or post-transcriptional levels. Broadly, this study will begin to identify the breadth and impact of small RNA targeting in existing transgenic lines and lay the foundation to explore how RNA interference in wild mosquitoes will respond to implementation of transgenic technologies to control mosquito-borne disease.