Germline transformation of the silkworm Bombyx mori L-using a piggyBac transposon-derived vector

We have developed a system for stable germline transformation in the silkwo rm Bombyx mori L. using piggyBac, a transposon discovered in the lepidopter an Trichoplusia ni. The transformation constructs consist of the piggyBac i nverted terminal repeats flanking a fusion of the B. mori cytoplasmic actin gene BmA3 promoter and the green fluorescent protein (GFP). A nonautonomou s helper plasmid encodes the piggyBac transposase. The reporter gene constr uct was coinjected into preblastoderm eggs of two strains of B. mori. Appro ximately 2% of the individuals in the G1 broods expressed GFP. DNA analyses of GFP-positive G1 silkworms revealed that multiple independent insertions occurred frequently, The transgene was stably transferred to the next gene ration through normal Mendelian inheritance. The presence of the inverted t erminal repeats of piggyBac and the characteristic TTAA sequence at the bor ders of all the analyzed inserts confirmed that transformation resulted fro m precise transposition events, This efficient method of stable gene transf er in a lepidopteran insect opens the way for promising basic research and biotechnological applications.