|Handler, Alfred - Al|
Submitted to: Proceedings of the National Academy of Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/7/1998
Publication Date: N/A
Citation: Handler, A.M., Mccombs, S.D., Fraser, M.J., Saul, S.H. 1998. The lepidopteran transposon vector, piggyBac, mediates germline transformation in the Mediterranean fruitfly. Proceedings of the National Academy of Sciences. 95:7520-7525. Interpretive Summary: The genetic manipulations of pest insects may reduce the need for pesticides for certain species. Therefore, the ability to achieve gene transfer in economicaaly important insects is a major goal of the Center for Medical, Agricultural and Veterinary Entomology, Gainesville, Florida. Development of this methodology depends upon the discovery and analysis of efficient and stable gene transfer vector systems. In this report ARS Scientists describe results from experiments designed to test the ability of the piggyBac vector from Trichoplusia ni, to mediate germline transformation in the Mediterranean fruit fly, Ceratitis capitata. Using a marker gene that results in the pigmentation of white-eye mutant flies, the piggyBac vector was shown to mediate efficient and stable transformation in the medfly. Gene transfer was verified by DNA hybridization tests and sequence analysis of the vector insertion site on the medfly chromosome. This is only the second gene transfer in an economically important insec, and is the first demonstration of a vector system from one insect order (Lepidoptera) mediating gene transfer in another insect order (Diptera). This is encouraging for the use of the piggyBac vector system in a wide variety of insect species. Potential use of piggyBac for improving the sterile insect technique for controlling fruit flies would be an important application.
Technical Abstract: The piggyBac (IFP2) transposable element from Trichoplusia ni was tested for gene transfer vector function as part of a bipartite vector:helper system in the Mediterranean fruit fly, Ceratitis capitata. A piggyBac vector marked with the medfly white gene, was coinjected with a normally regulated piggyBac transposase helper plasmid into white-eye host strain embryos. From 19 fertile G0 lines, three G1 sibling transformants were selected having dark-orange colored eyes. A single chromosomal piggyBac vector integration, stable for eight generations, was verified by DNA hybridization in all three lines. The integration was determined to be a piggyBac-mediated event by sequencing the insertion site junctions isolated by inverse PCR, yielding a TTAA duplication. Additional integrations not expressing the marker were detected, and a transformation frequency of 5- 10% is deduced. The efficient and stable transformation of the medfly with a lepidopteran vector indicates this vector might function in a wide range of insects.