ReMOT Control of mosquito transgenesis

描述（由申请人提供）：遗传操作是一种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或核酸内切酶递送到前胚盘胚胎。然而，胚胎显微注射在技术上是具有挑战性的，仅限于少数节肢动物类群，即使在优化的物种是低效的。因此，迫切需要开发简单的、许多研究人员可获得的并且通常与多种节肢动物物种相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到发育中的卵母细胞。当将果蝇卵黄蛋白1（DmYP 1）注射到卵黄发生前的成年雌性按蚊中时，其通过RME转导到种系中。我们表明，DmYP 1可以用于以100%的效率将货物如蛋白质或DNA转移到发育中的按蚊种系。我们将这种技术称为“受体介导的卵巢转运货物，或“ReMOT控制”，我们假设它可以用于将货物转移到按蚊种系中，以稳定和遗传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研究。第一个目标是使用ReMOT对照来特异性地删除斯氏按蚊生殖系中的基因。DmYP 1将与靶向GFP的转录激活因子样效应物核酸酶（TALEN）融合。在注射入GFP转基因蚊子后，通过荧光损失和测序鉴定缺失系。第二个目的是使用ReMOT Control通过将转座因子转导到蚊子生殖系中来创建转基因斯氏按蚊。DmYP 1将用于将含GFP的转座子转染到转基因转座酶表达蚊子中或与DmYP 1-转座酶融合酶共注射。将通过GFP功能获得鉴定转基因系;将通过PCR、测序和Southern印迹鉴定插入位点。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。

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.