Tn5 as an insect genomics tool

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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.