Selfish gene and genetic control of vector-borne diseases

描述（由申请人提供）：本提案旨在确定自私基因的潜力，即归巢内切酶，作为一种将基因分散和固定到自然蚊子种群中的方法，从而降低蚊子传播病原体（如登革热和疟疾的病原体）的媒介能力。在本提案的第一个目的，我们试图确定各种归巢内切酶的能力，以产生dsDNA断裂使用双质粒为基础的测定。这一试验已被设计成使成功切割归巢内切酶将导致负选择性标记的损失。将在培养的蚊子细胞和活的胚胎中进行基于质粒的检测。在第二个目标中，我们试图确定归巢内切酶从蚊子基因组中去除转基因的速率。在蚊子基因组中插入一个标记基因，两侧是归巢内切酶识别位点，然后引入一个归巢内切酶来切除标记。在基于质粒的测定和转基因切除测定中发现的归巢内切酶都具有功能，将为今后将归巢内切酶作为基因驱动方法的工作奠定基础。公共卫生相关性：这项工作是利用遗传学控制人类病原体的更大的、正在进行的战略的一部分。这种遗传控制策略建立在媒介能力降低/减弱将导致人类疾病相应减少/消除的假设之上，并基于三个要素：抗病原体效应基因的发展，将效应基因引入蚊子媒介的能力，以及将这些效应基因传播到野生种群的能力。前两个要素在过去十年中取得了重大进展，本建议旨在补充这项工作，使遗传控制战略能够成为现实，影响和改善因病媒传播疾病造成的公共卫生结果。

DESCRIPTION (provided by applicant): This proposal aims to determine the potential for selfish genes, AKA homing endonucleases, to serve as a method of dispersing and fixing genes into natural mosquito populations that reduce the vector competence of mosquitoes to transmit pathogens, such as the causative agents of dengue fever and malaria. In the first aim of this proposal we seek to determine the ability of various homing endonucleases to generate dsDNA breaks using a two-plasmid based assay. This assay has been designed so that successful cutting by the homing endonuclease will result in the loss of a negative selectable marker. Plasmid-based assays will be conducted in both cultured mosquito cells as well as in live embryos. In the second aim we seek to determine the rate at which homing endonucleases can excise a transgene from the mosquito genome. A marker gene flanked by homing endonuclease recognition sites will be inserted into the mosquito genome, followed by the introduction of a homing endonuclease which should excise the marker. Homing endonucleases found to be functional in both plasmid-based assays and transgene excision assays will serve as the foundation for future work at adapting homing endonucleases as a method of gene drive. Public health relevance: This work is part of a larger, ongoing strategy to control human pathogens using genetics. Such a genetic control strategy is founded on the hypothesis that reduced/ablated vector competence will result in a corresponding reduction/elimination of human disease, and is based on three elements: the development of anti-pathogen effector genes, the ability to introduce effector genes into mosquito vectors, and the ability to spread these effector genes into wild populations. The first two elements have seen substantial progress in the past decade, and this proposal is designed complement this work so genetic control strategies can become a reality in effecting and improving public health outcomes due to vector-borne diseases.