|Harrell, Robert - ENT DEPT, UNIV. OF FL|
Submitted to: Journal of Insect Biochemistry and Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 26, 2000
Publication Date: January 31, 2001
Citation: Handler, A.M., Harrell, R.A. 2001. Transformation of the Caribbean fruit fly with a piggyBac transposon vector marked with polyubiquitin-regulated GFP. Journal of Insect Biochemistry and Molecular Biology. 31:199-205. 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 tephritid fruit fly pests, piggyBac gene-transfer was tested in the Caribbean fruit fly, Anastrepha suspensa. A difficulty with this species, however, is that unlike the medfly, visible marking systems based on eye color mutations do not exist, thereby making selection of transgenic organisms difficult. We therefore tested a new marker system expressing the green fluorescent protein (GFP) gene from a jellyfish that is detected under ultraviolet light. The piggyBac vector and GFP marker was found to be an efficient gene transfer system. The ability to detect GFP externally in adult transgenic flies suggested that the same GFP marker could be used as a genetic marker to detect released flies in field studies. To optimize strains for GFP detection a new spectrofluorometric assay was developed.
Technical Abstract: Germline transformation was achieved in the Caribbean fruit fly, Anastrepha suspensa, using a piggyBac vector marked with an enhanced green fluorescent protein gene regulated by the Drosophila melanogaster polyubiquitin promoter. Four transgenic GO lines were selected exhibiting unambiguous GFP expression, and Southern hybridization indicated the presence of one to four integrations in each of the transgenic lines. Fluorescence was detectable throughout development, and in adults was most intense from the thoracic flight muscle. Although adult cuticle quenched fluorescence, GFP was routinely detectable in the thorax. A quantitative spectrofluorometric assay was developed for GFP fluorescence that indicated differing levels of fluorescence among the transgenic lines, suggesting some level of position effect variegation. These results are encouraging for the use of this marker system in insect species not amenable to mutation-based visible markers. Together with the piggyBac vector, a transformation system is presented that has the potential to be universally applicable in insect species.