The relationship between internal domain sequences of piggyBac and its transposition efficiency in BmN cells and Bombyx mori.

The piggyBac transposon, which includes terminal inverted repeat sequences and internal domain (ID) sequences, is widely used as a tool for insect transformation. To optimize this system for transgenic research on Bombyx mori, we examined the effects of the amount of the transposase plasmid and its ID sequences on the expression of green fluorescent protein (GFP). Four kinds of transposon plasmids, pB[A3GFP]-1 with the full length of ID sequences, pB[A3GFP]-2 having only the 3' ID sequence, pB[A3GFP]-3 without ID sequences, and pB[A3GFP]-4 containing 333 bp of the 5' ID sequence, and 179 bp of the 3' ID sequence were constructed with GFP as the marker. After transfecting these four plasmids into BmN cells, we analyzed the transfecting efficiency by comparing the GFP positive to negative cell ratio. Our results indicated that plasmid pB-4 got the highest ratio on the 22nd day. Moreover, the GFP positive to negative cell ratio increased with higher amount of transposase plasmid without overproduction inhibition. Furthermore, we injected three piggyBac transposon plasmids, pB[A3GFP]-1, pB[3xP3GFP]-3, and pB[3xP3RFP]-4 harboring different markers into preblastoderm stage eggs of B. mori, and found that the transformation efficiency of pB[3xP3RFP]-4 was 3.8 folds higher than pB[A3GFP]-1, whereas pB[3xP3GFP]-3 failed to produce transformants. Our results suggested that pB-4 may be one of the best piggyBac transposon plasmids currently available for germline transformation in B. mori.