| 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 bp in length, including a complex repeat configuration consisting of an internal repeat (IR), spacer, and terminal repeat (TR) at both ends, and a single open reading frame encoding the transposase. Excision assays and interplasmid transposition assays establish that piggyBac element is capable of precise excision and transposition in embryos of T. ni, Drosophila melanogaster and Aedes aegypti at relatively high frequencies. Excision assays of plasmids containing progressive deletions of the piggyBac internal sequence reveal that 5' and 3' IR, spacer, and TR configurations are sufficient for piggyBac precise excision when provided with transposase in trans. Interplasmid transposition assays of plasmids having varying lengths of intervening sequence between the piggyBac termini demonstrate a minimum of 55 bp sequence is required for optimal transposition, while lengths less than 40 bp result in a dramatic decrease in frequency. These results suggest that the piggyBac transposase might bind the termini simultaneously before cleavage take place, and/or that the formation of a transposition complex requires DNA bending between the two termini. Based on these results, we constructed a 702 bp cartridge having a minimum piggyBac 5' and 3' terminal region configuration separated by an optimal intervening sequence. Interplasmid transposition assays demonstrate that the minimum terminal configuration is sufficient to mediate transposition, and also verify that simply inserting this cartridge into an existing plasmid converts that plasmid into a non-autonomous piggyBac transposon. We also constructed a minimal piggyBac vector, pXL-Bac that contains an internal multiple cloning site sequence between the minimum terminal regions. These vectors will greatly facilitate the utilization of the piggyBac transposon in a wide range of hosts. The mutations of the piggyBac terminal region further confirm that IR, spacer and TR sequences are required for piggyBac "cut and paste" mobilization. |
