Precise excision of TTAA-specific lepidopteran transposons piggyBac (IFP2) and tagalong (TFP3) from the baculovirus genome in cell lines from two species of Lepidoptera
Excision precision of TTAA-specific transposons piggyBac and tagalong was untested. Both, tagged with lacZ and reinserted into the baculovirus genome, were screened as white-plaque revertants. Both excised precisely without their own transposase supplied in trans.
Key findings
- Fraser et al. demonstrated that piggyBac transposase catalyzes precise excision of transposon elements from baculovirus genomes without leaving sequence alterations at the donor site. Excision events occurred at high frequency in lepidopteran cell lines derived from Trichoplusia ni and Spodoptera frugiperda, restoring exact TTAA target sequences through a cut-and-paste mechanism.
- Molecular analysis of excision products revealed perfect reconstitution of TTAA sites at vacated donor loci, with no footprint mutations detected across multiple independent excision events. This precise reversal mechanism distinguishes piggyBac from other transposable elements that typically leave small deletions or insertions upon excision. The transposase-mediated process required functional inverted terminal repeats and occurred bidirectionally.
- Transposon excision frequency was enhanced by co-expression of piggyBac transposase, increasing from basal rates to >10% of cells in transient transfection assays. The tagalong (TFP3) element, sharing similar TTAA specificity, exhibited comparable precise excision properties. These findings established piggyBac as a reversible genetic tool with utility for transient and stable genomic modifications in insect systems.
Abstract
Transposon mutagenesis of baculoviruses provides an ideal experimental system for analysis of the movement of a unique family of mobile element identified from lepidopteran genomes. Members of this family of short-inverted-repeat elements are characterized by their extreme specificity for TTAA target sites. This report describes the analysis of excision events for two representatives of this family, tagalong (formerly TFP3) and piggyBac (formerly IFP2). These elements were tagged with a polyhedrin/lacZ reporter gene and inserted back into the virus genome either by homologous recombination or by transposition. Revertants were selected based on a white plaque phenotype. Both elements excise in a precise fashion from their positions in the baculovirus genome in either TN-368 cells or IPLB-SF21 AE cells. The precise excision of these elements in infected IPLB-SF21 AE cells occurs in the absence of either tagalong or piggyBac element encoded functions. The common characteristics of both insertion and excision for these elements provides further validation for their inclusion in a single family of unique transposons.