|Harrell, Robert - ENT DEPT, UNIV OF FL|
Submitted to: Insect Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 1, 1999
Publication Date: October 13, 1999
Citation: Handler, A.M., Harrell, R.A. 1999. Germline transformation of Drosophila melanogaster with piggyBac transposon vector. Insect Molecular Biology. 8:449-458. 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 fruit fly, Ceratitis capitata. In order to determine if this system can function as well in other dipteran insects, and perhaps insects in general, piggyBac gene-transfer was tested in Drosophila melanogaster. To optimize the frequency of gene-transfer as well as the selection of transgenic insects, we tested a new highly expressed "helper" gene and a new marker system expressing the green fluorescent protein (GFP) gene from a jellyfish. The piggyBac gene transfer system was found to be an efficient vector in Drosophila, and the new helper increased the gene-transfer frequency by eight-fold in comparison to the original helper. The GFP gene was found to be an effective marker, and should prove to be highly useful as a gene- transfer marker in agriculturally important insects.
Technical Abstract: Germline transformation of Drosophila melanogaster was attempted with the piggyBac gene transfer system from the cabbage looper moth, Trichoplusia ni. Using a self-regulated transposase helper, a transformation frequency of 1-3% per fertile GO was obtained, while use of an hsp70-regulated helper increased this frequency more than eight-fold. Transformation with a vector rmarked with white and green fluorescent protein (GFP) under polyubiquitin- nuclear localizing sequence regulation yielded 70 G1 transformants which all expressed GFP, but only 27 of these expressed eye pigmentation that would have allowed their selection based on white expression. piggyBac transformation in two distantly related dipteran species and the efficient expression of the gfp marker supports the potential use of this system in other dipterans, and perhaps insects in general