Tn5 as an insect genomics tool

描述（由申请人提供）：相关的更棘手的问题之一 在过去的20年里， 多年来一直是遗传转化技术的发展， 人类疟疾媒介冈比亚按蚊。遗传转化系统。 冈比亚和其他昆虫将用于研究昆虫/寄生虫的相互作用。 并开发新的基于遗传学的疾病控制策略 传输目前已有四种基于昆虫转座元件的基因载体 存在，但没有一个成功地用于产生转基因An。性追蚊和 在那些蚊子种类中没有一种是非常有效的， 来运作。来自细菌的Tn 5转座因子系统可以提供 成功转型的关键。冈比亚，因为它拥有 明显不同于现有的基于昆虫转座元件的系统。 首先，Tn 5系统在生物化学上得到了很好的表征，并已被修改 拥有1000倍于本地系统的活跃度。第二，积极 称为突触复合物或转座体TM的中间体可以在 并且可以直接引入待转化的细胞中。 引入这种活性中间体进一步提高了 转型使用一个超活性重组系统和能力， 在体外产生活性中间体可能是一个重要的关键 建立了一个转化系统。冈比亚将拥有真实的 实验室中的实用程序。初步数据显示，Tn 5在 昆虫生殖细胞在这里，Tn 5系统将在An进行测试。冈比亚因 结合将DNA引入发育中的蚊子的新方案， 胚胎20年来，创造转基因An的努力都失败了。冈比亚马克 这是一个高风险的项目，但它对医疗领域的影响， 昆虫学和控制疟疾传播的努力将是巨大的 如果成功的话。

DESCRIPTION (provided by applicant): One of the more vexing problems associated with the study of human diseases transmitted by insects over the last twenty years has been the development of genetic transformation technology for the human malaria vector, Anopheles gambiae. Genetic transformation systems for An. gambiae and other insects will be used to study insect/parasite interactions and to develop novel genetics-based strategies for controlling disease transmission. Four insect transposable element-based gene vectors currently exist but none have been successfully used to create transgenic An. gambiae and none are very efficient in those mosquito species in which they have been shown to function. The Tn5 transposable element system from bacteria may provide the key to successfully transforming An. gambiae because it has properties distinctly different from existing insect transposable element-based systems. First, the Tn5 system is biochemically well characterized and has been modified to have 1000 times the activity of the native system. Second, an active intermediate called a synaptic complex or Transposome TM can be produced in vitro and can be introduced directly into the cells to be transformed. Introducing this active intermediate further increases the efficiency of transformation. The use of a hyperactive recombination system and the ability to produce active intermediates in vitro may be an important key to the development of a transformation system for An. gambiae that will have real utility in the laboratory. Preliminary data show that Tn5 is functional in insect germ cells. Here the Tn5 system will be tested in An. gambiae in conjunction with a new protocol for introducing DNA into developing mosquito embryos. Two decades of failed efforts to create transgenic An. gambiae make this is a high risk project but one whose impact on the field of medical entomology and efforts to control the transmission of malaria will be enormous if it is successful.