AN. AGMBIAE GERM LINE TRANSFORMATION

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The long term objective of this project is to develop two different methods for integrating new DNA into the genome of An. Gambiae. The more immediate of these objectives is to develop a germ line transformation method based on one or more Class II transposons,, transposons that move dia a DNA intermediate through a cut-and-paste mechanism. We will initially explore plasmid-to-plasmid transpositional activity of 4 such transposons that have already been used to transform non-drosophilid insects: Hermes, Minos, Mos1 mariner, and piggyBac. These transposons that show transpositional mobility in An. Gambiae embryos (this has already been documented for Hermes) will be examined in more detail in such assays in an effort to optimize plasmid- to-plasmid transposition by varying helper-to-donor plasmid ratios. At the same time, we will be conducting a gamma-ray mutagenesis screen of An. Gambiae in order to produce strains with a mutations in one or both of two genes that encode enzymes important for ommochrome eye pigment synthesis: tryptophan oxygenase (vermillion in D. melanogaster) and kynurenine hydroxylase (cinnabar in D. melanogaster). The resulting mutant strain(s) will then be the target of transformation experiments with constructs based on the most promising of the above Class II transposons. We will use wild type tryptophan oxygenase or kynurenine hydroxylase receptor constructs that already exit and have been validated in other Diptera (Ae. Aegypti and D. melanogaster). Finally, when a functional An. Gambiae transformation method based on a Class II transposons is available, we will undertake to develop a Cre/lox target site-specific recombination system for "docking" large fragments of DNA (in excess of 20 kb) in to the An. Gambiae genome. In this latter aim, we will be guided by the results of Drs. Lucy and Peter Cherbas at Indiana University who are currently developing such a method for D. melanogaster and Anthony A. James at UC Irvine who is doing similar work with Ae. Aegypti.