Small RNA interactions with transgenes in genetically modified mosquito lines

小RNA与转基因蚊子品系中转基因的相互作用

• 批准号: 10654368
• 负责人: Vanessa Macias
• 金额: $ 46.64万
• 依托单位: UNIVERSITY OF NORTH TEXAS
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10654368
• 关键词: Aedes; Anopheles Genus; Arboviruses; Bacteriophages; Behavior; Biogenesis; Biological Assay; Biological Models; Biology; Complex; Culicidae; DNA; DNA Transposable Elements; Data; Dedications; Disease Vectors; Docking; Elements; Engineering; Fluorescence; Foundations; Future; Gene Expression Regulation; Gene Transfer Techniques; Genes; Genetic; Genetic Transcription; Genome Stability; Genome engineering; Genomics; Goals; Human; Knowledge; Laboratories; Libraries; Molecular; Molecular Biology; Molecular Genetics; Mosquito Control; Mosquito-borne infectious disease; Nuclear Localization Signal; Organism; Outcome; Output; Pathway interactions; Pattern; Phenotype; Plasmids; Population; Post-Transcriptional Regulation; Proteins; Protocols documentation; Public Health; Publishing; RNA; RNA Interference; Regulation; Reporter; Reporting; Repression; Research Personnel; Residual state; Resources; Role; Sequence Homology; Site; Small Interfering RNA; Small RNA; Target Populations; Technology; Testing; Time; Tissues; Transcript; Transgenes; Transgenic Organisms; Vector-transmitted infectious disease; Viral; Virus; Visual; Work; arm; bioinformatics resource; data integration; design; design and construction; disorder control; gene repression; genetic approach; improved; innovation; interdisciplinary approach; interest; knock-down; loss of function; mRNA Expression; model organism; mosquito-borne pathogen; offspring; piRNA; posttranscriptional; protein expression; restoration; sensor; success; synthetic construct; transcriptome sequencing; transgene expression; vector; vector control

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.

项目总结： 利用转基因蚊子应用于控制媒介传播疾病的策略依赖于 合成转基因的活性，可以编码新的表型，如灭蚊或阻断病毒 表型。观察到转基因可能会意外丧失功能，这与对 通过一种名为piRNA途径的RNA干扰机制将外源DNA转化为DNA。初步的小RNA 埃及伊蚊和斯氏按蚊功能缺失转基因品系的序列分析 表型揭示了不同程度的siRNAs和piRNAs对转基因的靶向作用。这样做的目的是 研究是确定蚊子中潜在的针对转基因的小RNA活性，并直接测试 这种小RNA可以抑制转基因活性。第一个目标是通过富集法检测siRNA和piRNA。 以及蚊子发育阶段和与转基因活性相关的解剖组织中的小RNA测序 和继承权都在Ae.埃及斑潜蝇和An.并整合这些数据 以及发表的转基因序列进入生物信息学资源，将支持假说检验 其他实验室和环境。这方面的一个主要成果将是一个资源，可以让研究人员设计 构造以避免小RNA识别。第二个目标是直接测试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感应器菌株来实现这一点 背景，Ae.埃及斑潜蝇和An.斯特芬西。该菌株将含有一个带有荧光标记的基因组拷贝 设计成可被特定的小RNA检测到。如果小RNA是抑制性的，那么它们的后代 传感器菌株和被测菌株会导致荧光减弱。如果siRNA和piRNAs负责 对于这种表型，siRNA和piRNA生物发生蛋白DICER2或Zucchini将分别被击倒 拯救荧光蛋白的表达。来自这个杂交的活性也将通过测试丰富的 记者抄本以及通过测序产生的针对记者的小RNA，这可以提供 关于抑制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 开始识别小RNA靶向在现有转基因品系中的广度和影响，并奠定 基金会探索野生蚊子的RNA干扰将如何应对转基因的实施 控制蚊媒疾病的技术。