|Perera, Omanththage - UNIV OF FL, GAINESVILLE|
|Harrell Ii, Robert - UNIV OF FL, GAINESVILLE|
Submitted to: Insect Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 19, 2002
Publication Date: August 1, 2002
Citation: Perera, O.P., Harrell Ii, R.A., Handler, A.M. 2002. Germ-line transformation of the South American malaria vector, Anopheles albimanus, with a piggyBac/EGFP transposon vector is routine and highly efficient. Insect Molecular Biology. 11:291-297. Interpretive Summary: The ability to achieve gene transfer in economically important insects is a major goal of our laboratory at the CMAVE. Development of this methodology depends upon the discovery and analysis of efficient and stable gene transfer vector systems. Previously, the piggyBac vector from Trichoplusia ni was found to mediate germline transformation in the Mediterranean and Caribbean fruit flies. In order to determine if this system can function a well in other dipteran insects, piggyBac gene-transfer was tested in the South American malaria vector, Anopheles albimanus. We selected transformed insects in four experiments at a relatively high frequency. Similar to previous experiments in Anopheles mosquitoes, adult survival and fertility was low, but the transformation frequencies were among the highest observed with the piggyBac vector. This is encouraging for the further use of the piggyBac vector system in a wide range of dipteran insects.
Technical Abstract: Stable and efficient germ-line transformation was achieved in the South American malaria vector, Anopheles albimanus, using a piggyBac vector marked with an enhanced green fluorescent protein gene regulated by the Drosophila melanogaster polyubiquitin promoter. Transgenic mosquitoes were selected from four independent experiments at frequencies ranging from 20 to 43% per fertile Go. Fluorescense was observable throughout the body of larvae and pupae, and abdominal segments of adults. Transgenic lines analyzed by Southern hybridization had one to six germ-line integrations, with most lines having three or more integrations. Hybridized vector fragments and insertion site sequences were consistent with precise piggyBac-mediated integrations, although this was not verified for all lines. The piggyBac/PUbn1sEGFP vector appears to be a robust transformation system for this anopheline species, in contrast to the use of piggyBac in An. gambiae. Further tests are needed to determine if differences in anopheline transformation efficiency are due to the marker systems or to organismal or cellular factors specific to the species.