Abstract
BackgroundTransposons are useful tools for creating transgenic organisms, insertional mutagenesis, and genome engineering. TcBuster, a novel hAT-family transposon system derived from the red flour beetle Tribolium castaneum, was shown to be highly active in previous studies in insect embryoes.Methodology/Principal FindingsWe tested TcBuster for its activity in human embryonic kidney 293 (HEK-293) cells. Excision footprints obtained from HEK-293 cells contained small insertions and deletions consistent with a hAT-type repair mechanism of hairpin formation and non-homologous end-joining. Genome-wide analysis of 23,417 piggyBac, 30,303 Sleeping Beauty, and 27,985 TcBuster integrations in HEK-293 cells revealed a uniquely different integration pattern when compared to other transposon systems with regards to genomic elements. TcBuster experimental conditions were optimized to assay TcBuster activity in HEK-293 cells by colony assay selection for a neomycin-containing transposon. Increasing transposon plasmid increased the number of colonies, whereas gene transfer activity dependent on codon-optimized transposase plasmid peaked at 100 ng with decreased colonies at the highest doses of transposase DNA. Expression of the related human proteins Buster1, Buster3, and SCAND3 in HEK-293 cells did not result in genomic integration of the TcBuster transposon. TcBuster, Tol2, and piggyBac were compared directly at different ratios of transposon to transposase and found to be approximately comparable while having their own ratio preferences.Conclusions/Significance TcBuster was found to be highly active in mammalian HEK-293 cells and represents a promising tool for mammalian genome engineering.
Highlights
Transposon technology has been harnessed for genome engineering for the creation of transgenic organisms [1], cancer gene discovery by insertional mutagenesis [2], and in pre-clinical gene transfer experiments by inserting genes into the genomes of the somatic cells of living organisms [3]
Transposon systems are known to differ in their target site preference and these differences have been exploited for expanding cancer gene discovery [13]
The transposons used in this study each contained an identical cassette encoding for a drug-resistance gene, so that the transfected cells could be selected with either neomycin or blasticidin to select for the sustained expression of the transgene
Summary
Transposon technology has been harnessed for genome engineering for the creation of transgenic organisms [1], cancer gene discovery by insertional mutagenesis [2], and in pre-clinical gene transfer experiments by inserting genes into the genomes of the somatic cells of living organisms [3]. While the most studied transposon for these applications has been Sleeping Beauty, a reconstructed Tc1/mariner-type transposon from fish [4], others such as Frog Prince [5], Tol2 [6], and piggyBac [7] have proven to be highly active in diverse organisms and applications. They share a similar overall mechanism of ‘‘cut and paste’’ of the delivered transposon DNA, each transposon system has its own characteristics. TcBuster, a novel hAT-family transposon system derived from the red flour beetle Tribolium castaneum, was shown to be highly active in previous studies in insect embryoes
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