title: Comparative analysis of the recently discovered hAT transposon TcBuster in human cells.
abstract: BACKGROUND
  Transposons 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.
  
  
  RESULTS
  We 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
  TcBuster was found to be highly active in mammalian HEK-293 cells and represents a promising tool for mammalian genome engineering.
authors.affiliation: Department of Medicine, Division of Nephrology, Baylor College of Medicine, Houston, Texas, USA.
authors.forename: Lauren E
authors.initials: LE
authors.lastname: Woodard
authors.forename: Xianghong
authors.initials: X
authors.lastname: Li
authors.forename: Nirav
authors.initials: N
authors.lastname: Malani
authors.forename: Aparna
authors.initials: A
authors.lastname: Kaja
authors.forename: Robert H
authors.initials: RH
authors.lastname: Hice
authors.forename: Peter W
authors.initials: PW
authors.lastname: Atkinson
authors.forename: Frederic D
authors.initials: FD
authors.lastname: Bushman
authors.forename: Nancy L
authors.initials: NL
authors.lastname: Craig
authors.forename: Matthew H
authors.initials: MH
authors.lastname: Wilson
mesh_terms: D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004251:DNA Transposable Elements / Q000235:genetics*; D018014:Gene Transfer Techniques; D005818:Genetic Engineering / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D054643:INDEL Mutation / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods*; D010957:Plasmids / Q000235:genetics; D019895:Transposases / Q000378:metabolism; D014231:Tribolium / Q000235:genetics*
chemical_list: D017931:DNA Primers; D004251:DNA Transposable Elements; D019895:Transposases