ReMOT Control of mosquito transgenesis

描述(由申请人提供)：基因操作是解决具有重要医学意义的节肢动物研究问题的一种强有力的技术。目前的方法依赖于通过胚胎显微注射将DNA或核酸内切酶输送到前胚层胚胎。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分类群，即使在优化的物种中也效率低下。因此，迫切需要开发一种简单的、许多研究人员都能接触到的节肢动物遗传操作方法，并且通常与多种节肢动物物种兼容。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到发育中的卵母细胞。将果蝇黑腹果蝇卵黄蛋白1(DmYP1)注射到卵黄形成前期的雌性按蚊体内，通过RME将其导入生殖系。我们证明，DmYP1可以100%的效率将蛋白质或DNA等货物转导到发育中的按蚊生殖系。我们将这项技术称为受体介导的卵巢货物转导，或“远程控制”，我们假设可以用来将货物转导到按蚊生殖系，以稳定和可遗传地编辑蚊子染色体遗传序列。这一假设将通过两个具体目标进行调查。第一个目标是使用远程控制专门删除斯氏按蚊种系中的基因。DmYP1将与靶向GFP的转录激活物样效应核酸酶(TALEN)融合。注射入GFP转基因蚊子后，通过荧光损失和测序鉴定缺失品系。第二个目标是使用Remot Control通过将转座元件转导到蚊子生殖系中来创造转基因斯氏按蚊。DmYP1将被用来将含有GFP的转座子转导到表达转座酶的转基因蚊子中，或者与DmYP1转座酶融合酶共注射。转基因品系将通过GFP功能获得鉴定；插入位点将通过聚合酶链式反应、测序和Southern印迹鉴定。一旦得到优化，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、灵活的基因操作。

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.