Hermes Element Transposition in Mosquitoes

蚊子中的赫耳墨斯元素转位

• 批准号: 7010012
• 负责人: PETER William ATKINSON
• 金额: $ 41.79万
• 依托单位: UNIVERSITY OF CALIFORNIA RIVERSIDE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-30 至 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7010012
• 关键词: Aedes; animal population genetics; arthropod genetics; dimer; disease vectors; genetically modified animals; laboratory mouse; site directed mutagenesis; transfection /expression vector; transposon /insertion element

DESCRIPTION (provided by the applicant): The long-term goal of this project is to understand the behavior of introduced transposable elements in transgenic mosquitoes that are vectors of human disease. It is the central proposition of the research described herein that the ability of a transposable element to be used both as an efficient genetic tool in the laboratory in mosquitoes, as well as an effective "gene driving mechanism" of beneficial effector genes in the field is determined by both how the transposable element functions, and how it interacts with the new host mosquito genome. This proposition will be examined using the Hermes transposable element, which is a member of the hAT family of transposable elements, and which is an effective transformation vector of the mosquito, Aedes aegypti. The Specific Aims of this proposal are thus: 1/ to purify and characterize the Hermes transposase, 2) to understand the structure: function relationships of the Hermes element, 3) to examine interactions of the Hermes transposase, and 4) to isolate hyperactive mutants of Hermes transposase and to test them for hyperactivity in mosquitoes. Purified biologically active Hermes transposase will be used to determine the optimal physical and biological parameters for Hermes element excision and transposition and for the role that host factors may play in Hermes element transposition in mosquitoes. Regions of the both the transposase and the element, such as the inverted terminal repeats and adjacent conserved motifs, that are required for transposition will be identified and, using mutagenesis, their precise roles in this process determined. The role of circular forms of the Hermes element, which we have identified to be present in transgenic insects containing autonomous forms of Hermes element will be investigated. Finally, both eukaryotic and prokaryotic based expression systems, both of which produce active Hermes transposase, will be used to identify hyperactive forms of the Hermes element system that will then be tested for elevated levels of activity in Ae. aegypti. To date we know little about how the transposable elements used to genetically transform mosquitoes either work or behave in mosquito genomes. This now constitutes the most significant bottleneck to the extension of transposable element-based technologies into medically important mosquito species. This technology will complement existing approaches to the control of mosquito-borne disease control and will provide new avenues with which to approach urgent problems arising from the increase in the geographical distribution of these devastating diseases which impact both human health and national economies. This new technology will enable mosquito genes required for pathogen transmission to be identified and isolated, will improve the efficiency with which transgenic mosquito strains can be identified, and will also provide important data concerning the likelihood that genes that prevent pathogen transmission can be spread through mosquito populations by transposable elements.

描述（由申请人提供）：该项目的长期目标是了解作为人类疾病载体的转基因蚊子中引入的转座因子的行为。本文所述的中心命题是，转座元件在实验室中作为蚊子的有效遗传工具，以及在实地中作为有益效应基因的有效“基因驱动机制”的能力，取决于转座元件如何发挥作用，以及它如何与新的宿主蚊子基因组相互作用。这一命题将使用Hermes转座因子进行检验，Hermes转座因子是hAT转座因子家族的成员，是埃及伊蚊（Aedes aegypti）的有效转化载体。因此，本研究的具体目的是：1)纯化和表征Hermes转座酶；2)了解Hermes元件的结构和功能关系；3)研究Hermes转座酶的相互作用；4)分离Hermes转座酶的高活性突变体并测试它们在蚊子中的高活性。纯化的生物活性Hermes转座酶将用于确定Hermes元件切除和转座的最佳物理和生物参数，以及宿主因素在蚊子Hermes元件转座中可能发挥的作用。转座酶和元件的区域，如倒置末端重复序列和邻近的保守基序，是转座所需的，将被识别，并使用诱变，确定其在这一过程中的精确作用。圆形形式的赫尔墨斯元素的作用，我们已经确定存在于转基因昆虫含有自主形式的赫尔墨斯元素将被调查。最后，真核生物和原核生物的表达系统都能产生活性Hermes转座酶，将用于鉴定Hermes元件系统的超活性形式，然后测试Ae中活性水平的升高。蚊。