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）：基因操纵是解决医学重要性节肢动物研究问题的有力技术。当前的方法依赖于通过胚胎微分注射将DNA或核酸内切酶传递给蓝细胞胚胚。但是，胚胎的微注射在技术上具有挑战性，仅限于少数节肢动物分类群，即使在优化的物种中也效率低下。因此，至关重要的是开发用于节肢动物遗传操作的方法，这些方法简单，对于许多研究人员来说都是可以访问的，并且通常适合各种节肢动物物种。在卵子发生过程中，昆虫通过受体介导的内吞作用（RME）将蛋黄蛋白前体转移到发育中的卵母细胞。当果蝇果蝇蛋白蛋白1（DMYP1）被注入前毛状成年雌性蚊子蚊时，它会被RME转导为种系。我们表明，DMYP1可用于将具有100％效率的蛋白质或DNA等货物转导至发育中的肛门种系。我们将这种技术称为“受体介导的卵巢转导货物或“远程控制”，我们假设可以将其用于将货物转换为蚊帐种系，以稳定且可遗传的编辑蚊子染色体遗传序列。 Stephensi。将DMYP1融合到靶向GFP之后的转录激活效应子核酸酶（TALES）。 DMYP1将用于将含GFP的转座子转置转基因转座酶表达蚊子或与DMYP1-转座酶融合酶共同注射。插入位点将通过PCR，测序和Southern印迹来识别。一旦进行了优化，远遥控制将极大地改变分子昆虫学研究的景观，从而可以轻松，灵活地对各种载体节肢动物和非模型物种进行柔软的遗传操纵。