Transformation of the Caribbean fruit fly, Anastrepha suspensa, with a piggyBac vector marked with polyubiquitin-regulated GFP.
Germ-line 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 G(0) lines were selected exhibiting unambiguous GFP expression. Southern hybridization indicated the presence of one to four integrations in each of the transgenic lines with two integrations verified as piggyBac-mediated by sequencing their insertion sites. 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/suppression. 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.
Handler
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Center for Medical, Agricultural, and Veterinary Entomology, Agricultural Research Service, US Department of Agriculture, Gainesville, FL 32608, USA. handler@nersp.nerdc.ufl.edu
Harrell
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D000818:Animals; D030801:Animals, Genetically Modified; D001665:Binding Sites; D001704:Biopolymers / Q000235:genetics*; D015139:Blotting, Southern / Q000379:methods; D004175:Diptera; D015870:Gene Expression; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics*; D025821:Polyubiquitin; D011401:Promoter Regions, Genetic; D014170:Transformation, Genetic; D014452:Ubiquitins / Q000235:genetics*
D001704:Biopolymers; D008164:Luminescent Proteins; D014452:Ubiquitins; D025821:Polyubiquitin; D049452:Green Fluorescent Proteins