The minimum internal and external sequence requirements for transposition of the eukaryotic transformation vector piggyBac

The piggyBac element from Trichoplusia ni is recognized as a useful vector for transgenesis of a wide variety of species. This transposable element is 2472 by in length, and has a complex repeat configuration consisting of an internal repeat (IR), spacer, and terminal repeat (TR) at both ends, and a single ORF encoding the transposase. Excision assays performed in microinj ected T. ni embryos using plasmids deleted for progressively larger portion s of the piggyBac internal sequence reveal that the 5' and 3' IR, spacer, a nd TR configuration is sufficient for precise excision of piggyBac when tra nsposase is provided in trans. Interplasmid transposition assays using plas mids carrying varying lengths of intervening sequence between the piggyBac termini in T. ni demonstrate that a minimum of 55 by of intervening sequenc e is required for optimal transposition, while lengths less than 40 by resu lt in a dramatic decrease in transposition frequency. These results suggest that the piggyBac transposase may bind both termini simultaneously before cleavage can occur, and/or that the formation of a transposition complex re quires DNA bending between the two termini. Based on these results we const ructed a 702-by cartridge with minimal piggyBac 5' and 3' terminal regions separated by an intervening sequence of optimal length. Interplasmid transp osition assays demonstrate that the minimal terminal configuration is suffi cient to mediate transposition, and also verify that simply inserting this cartridge into an existing plasmid converts that plasmid into a non-autonom ous piggyBac transposon. We also constructed a minimal piggyBac vector, pXL -Bac, that contains an internal multiple cloning site sequence between the minimal terminal regions. These vectors should greatly facilitate the utili zation of the piggyBac transposon in a wide range of hosts.