piggyBac-based insertional mutagenesis in Tribolium castaneum using donor/helper hybrids

We describe an efficient method for generating new piggyBac insertions in the germline of F-1 hybrid Tribolium castaneum derived from crosses between transgenic helper and donor strains. Helper strains carried single Minos elements encoding piggyBac transposase. The donor strain carried a single piggyBac element inserted into an actin gene, expanding the eye-specific, 3xP3-EGFP (enhanced green fluorescent protein) reporter expression domain to include muscle. Remobilization of the donor element is accompanied by loss of muscle fluorescence but retention of eye fluorescence. In a pilot screen, the piggyBac donor was remobilized in 84% of the hybrid crosses, generating hundreds of new lethal, enhancer-trap, semisterile and other insertions. The jumpstarter system described herein makes genome-wide, saturation insertional mutagenesis a realistic goal in this coleopteran species.