ReMOT Control of mosquito transgenesis

蚊子转基因的 ReMOT 控制

基本信息

批准号：
8683890
负责人：
Jason L Rasgon
金额：
$ 22.43万
依托单位：
PENNSYLVANIA STATE UNIVERSITY, THE
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-02-05 至 2016-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8683890
关键词：
Address Adult Anopheles Genus Arthropod Vectors Arthropods Culicidae DNA DNA Binding Domain DNA Transposable Elements Drosophila melanogaster Egg Yolk Proteins Embryo Engineering Entomology Enzymes FUS-1 Protein Female Fluorescence Gene Deletion Gene Transfer Techniques Genes Genetic Human Injection of therapeutic agent Insecta Malaria Mediating Medical Methods Microinjections Molecular Mosquito Control Oocytes Oogenesis Ovary Protein Precursors Proteins Research Research Personnel Site Southern Blotting Taxon Techniques Transcription Coactivator Transgenic Organisms Transposase Vector-transmitted infectious disease Western Blotting arthropod genetics endonuclease enhanced green fluorescent protein flexibility gain of function genetic manipulation interest knockout gene novel nuclease offspring public health relevance receptor receptor mediated endocytosis research study restriction enzyme vector

项目摘要

DESCRIPTION (provided by applicant): Genetic manipulation is a powerful technique for addressing research questions in arthropods of medical importance. Current approaches rely upon delivering DNA or endonucleases to preblastoderm embryos via embryonic microinjection. However, embryonic microinjection is technically challenging, is limited to a small number of arthropod taxa, and is inefficient even in optimized species. As such, there is a critical need to develop methods for arthropod genetic manipulation that are simple, accessible for many researchers and generally compatible for a large variety of arthropod species. During oogenesis, insects transfer yolk protein precursors to developing oocytes by receptor-mediated endocytosis (RME). When Drosophila melanogaster yolk protein 1 (DmYP1) is injected into pre-vitellogenic adult female Anopheles mosquitoes, it is transduced into the germline by RME. We show that DmYP1 can be used to transduce cargo such as protein or DNA with 100% efficiency to the developing Anopheles germline. We term this technique "Receptor-Mediated Ovary Transduction of Cargo, or "ReMOT Control", which we hypothesize can be used to transduce cargo into the Anopheles germline for stable and heritable editing of the mosquito chromosomal genetic sequence. This hypothesis will be investigated by two specific Aims. The first aim is to use ReMOT Control to specifically delete genes in the germline of Anopheles stephensi. DmYP1 will be fused to transcription activator-like effector nucleases (TALENs) targeting GFP. After injection into GFP-transgenic mosquitoes, deletion lines will be identified by loss of fluorescence and sequencing. The second aim is to use ReMOT Control to create transgenic Anopheles stephensi by transduction of transposable elements into the mosquito germline. DmYP1 will be used to transduce GFP-containing transposons into transgenic transposase-expressing mosquitoes or co-injected with DmYP1-transposase fusion enzymes. Transgenic lines will be identified by GFP gain-of- function; insertion sites will be identified by PCR, sequencing and southern blot. Once optimized, ReMOT Control will dramatically change the landscape of molecular entomology research, allowing easy, flexible genetic manipulation of a wide variety of vector arthropods and non-model species.

描述（由申请人提供）：基因操作是解决具有医学重要性的节肢动物研究问题的强大技术。目前的方法依赖于通过胚胎显微注射将 DNA 或核酸内切酶递送至前胚层胚胎。然而，胚胎显微注射在技术上具有挑战性，仅限于少数节肢动物类群，并且即使在优化的物种中效率也很低。因此，迫切需要开发简单、许多研究人员易于使用并且通常与多种节肢动物物种兼容的节肢动物遗传操作方法。在卵子发生过程中，昆虫通过受体介导的内吞作用（RME）将卵黄蛋白前体转移至发育中的卵母细胞。当果蝇卵黄蛋白 1 (DmYP1) 被注射到卵黄发生前的成年雌性按蚊体内时，它会通过 RME 转导到种系中。我们证明，DmYP1 可用于将蛋白质或 DNA 等货物转导至发育中的按蚊种系，效率为 100%。我们将这种技术称为“受体介导的货物卵巢转导”，或“ReMOT 控制”，我们假设该技术可用于将货物转导到按蚊种系中，以对蚊子染色体基因序列进行稳定和可遗传的编辑。该假设将由两个具体目标。第一个目标是使用 ReMOT Control 特异性删除 DmYP1 种系中的基因，使其与转录融合。靶向 GFP 的类激活子效应核酸酶 (TALEN)。注射到 GFP 转基因蚊子中后，将通过荧光缺失和测序来鉴定缺失系。第二个目标是使用 ReMOT Control 通过将转座元件转导到转基因蚊子中来创建转基因按蚊。 DmYP1 将用于将含有 GFP 的转座子转导至表达转基因转座酶的蚊子中或与DmYP1-转座酶融合酶将通过 GFP 功能获得来鉴定；插入位点将通过 PCR、测序和 Southern blot 来鉴定。一旦优化，ReMOT Control 将极大地改变分子昆虫学研究的格局，允许对各种媒介节肢动物和非模式物种进行简单、灵活的遗传操作。