Abstract
The piggyBac transposon, originating in the genome of the Lepidoptera Trichoplusia ni, has a broad host range, making it useful for the development of a number of transposon-based functional genomic technologies including gene vectors, enhancer-, gene- and protein-traps. While capable of being used as a vector for the creation of transgenic insects and insect cell lines, piggyBac has very limited mobility once integrated into the genome of the yellow fever mosquito, Aedes aegypti. A transgenic Aedes aegypti cell line (AagPB8) was created containing three integrated piggyBac elements and the remobilization potential of the elements was tested. The integrated piggyBac elements in AagPB8 were transpositionally silent in the presence of functional transposase, which was shown to be capable of catalyzing the movement of plasmid-borne piggyBac elements in the same cells. The structural integrity of one of the integrated elements along with the quality of element-flanking DNA, which is known to influence transposition rates, were tested in D. melanogaster. The element was found to be structurally intact, capable of transposition and excision in the soma and germ-line of Drosophila melanogaster, and in a DNA sequence context highly conducive to element movement in Drosophila melanogaster. These data show that transpositional silencing of integrated piggyBac elements in the genome of Aedes aegypti appears to be a function of higher scale genome organization or perhaps epigenetic factors, and not due to structural defects or suboptimal integration sites.
Highlights
DNA transposons are abundant and influential residents of many genomes and have been harnessed as gene vectors and functional genomics tools because their high mobility and simple structures permit them to be modified and manipulated [1,2]
Like a number of other eukaryotic DNA transposons, piggyBac can transpose under some conditions in a host-independent manner making it an excellent platform upon which to build gene vectors and other functional genomics tools [13,14,15,16,17]. piggyBac has been used as a gene vector for the creation of growing number of transgenic insects of a wide range of species and in a few of those species more sophisticated piggyBacbased forward genetics and functional genomics tools have been successfully developed [18]
Cell line AagPB8 was used for all experiments and contains two integrated copies of pBac:Act5cHyg:ie1EGFP resulting from canonical cut-and-paste transposition of piggyBac and a third element associated with the integration of a copy of the plasmid pBac:Act5cHyg:ie1EGFP (Table 1)
Summary
DNA transposons are abundant and influential residents of many genomes and have been harnessed as gene vectors and functional genomics tools because their high mobility and simple structures permit them to be modified and manipulated [1,2]. We report that post-integration silencing of piggyBac mobility occurs in Ae. aegypti cells in vitro, that silencing occurs in the presence of functional transposase and that when silent elements are removed from the genome with approximately one kilobase of flanking chromosomal DNA and inserted into the genome of D. melanogaster they are fully active.
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