Forward and Reverse Genetics in Mosquitoes Using an RNA Virus

描述（由申请人提供）：本提案旨在利用RNA病毒的强大复制，使用正向和反向遗传方法研究病媒蚊子的基因功能。在第一个目标中，编码病毒复制酶的RNA病毒基因组的一部分将以组织特异性方式表达为来自蚊子基因组的转基因，并将原生蚊子基因序列整合到病毒基因组中。随着病毒复制的进行，RNA干扰应同时诱导到病毒RNA和原生蚊子基因。我们预计，由于病毒复制酶介导的扩增步骤，影响反向重复序列直接转录的核转录率的线性变异性应该被消除或大幅减少，这种变异性可能由转基因插入位点、营养状况或其他因素引起。这将提高基于RNAi的蚊子反向遗传实验的强度和效率，并简化RNAi敲除表型的解释。在第二个目标中，我们寻求开发一种参与RNA干扰途径的蚊子基因的正向遗传筛选。虽然最明显的RNAi基因已经被表征，但可能有许多关键成分仍然未知。提出了一种基于文库的重组病毒系统，当且仅当特定的重组病毒含有一部分参与RNAi的基因时，该系统将支持病毒复制。公共卫生相关性。埃及伊蚊和冈比亚按蚊造成了包括登革出血热和疟疾在内的巨大疾病负担。开发更精确、更有效和更稳健地中断蚊子基因功能的方法对于了解这些媒介的生物学以及了解病原体传播机制至关重要。了解参与先天抗病毒免疫的基因将有助于更好地了解媒介能力，并对引入的病原体的传播潜力进行建模，无论是自然的还是通过生物恐怖主义。

DESCRIPTION (provided by applicant): This proposal aims to utilize the robust replication of an RNA virus to study gene function in disease vector mosquitoes using both forward and reverse genetic approaches. In the first aim, a portion of an RNA viral genome encoding the viral replicase will be expressed as a transgene from the mosquito genome in a tissue-specific manner, with a native mosquito gene sequence incorporated into the virus genome. As viral replication proceeds, RNA interference should be induced to both the viral RNA and the native mosquito gene. We expect that because of the viral replicase-mediated amplification step, linear variability in nuclear transcription rates, which affects the direct transcription of inverted repeat sequences and can be caused by transgene insertion sites, nutritional status or other factors, should be eliminated or substantially reduced. This should increase both the strength and the efficiency of RNAi-based reverse genetic experiments in mosquitoes as well as simplify the interpretation of RNAi knockout phenotypes. In the second aim, we seek to develop a forward genetic screen for mosquito genes involved in the RNA interference pathway. While the most obvious RNAi genes have been characterized, there are likely many critical components which remain unknown. A library-based recombinant virus system is proposed which will support virus replication if and only if a particular recombinant virus contains a portion of a gene involved in RNAi. Public Health Relevance. The mosquitoes Aedes aegypti and Anopheles gambiae are responsible for an enormous disease burden including dengue hemorrhagic fever and malaria. Developing methods to more precisely, more efficiently and more robustly interrupt mosquito gene function is critical to understanding the biology of these vectors, as well as understanding mechanisms of pathogen transmission. Understanding the genes involved in innate anti-viral immunity will allow better understanding of vector competence and modeling of transmission potential for introduced agents, either natural or through bioterrorism.