STABLE TRANSFORMATION OF MOSQUITO CELL-LINES USING A HSP--NEO FUSION GENE

Mosquito cell culture transfection will allow the advancement of genet ic studies of these important disease-transmitting insects. Towards th is end, we report the generation of stably transformed Aedes aegypti M os20 cells using a plasmid construct containing the Tn5 neo gene, the Drosophila melanogaster hsp70 promoter, an SV40 intron and poly adenyl ation sequence, and a pBR322 backbone. The apparent frequency of trans fection, as measured by transient resistance of cell colonies to Genet icin (G418), ranged between 1 x 10(-4) and 1 x 10(-5), whereas the mea n frequency of transformation, as assessed by establishment of cloned lines, was 3.3 x 10(-6). The stable cell lines display typical charact eristics common to mammalian cell lines transformed with plasmids, inc luding stable resistance to G418 after removal of selection, and co-tr ansformation with unlinked plasmids. However, in contrast to the repor t of transformation of Ae. albopictus cells [Monroe et al., Proc. Natl . Acad. Sci. USA 89 (1992) 5725-5729], the plasmids within transformed Ae. aegypti cells have a wide range of copy number (3 to 5000), are e xtensively rearranged, and are only found integrated into the chromoso me.