title: Active recombinant Tol2 transposase for gene transfer and gene discovery applications.
abstract: BACKGROUND
  The revolutionary concept of "jumping genes" was conceived by McClintock in the late 1940s while studying the Activator/Dissociation (Ac/Ds) system in maize. Transposable elements (TEs) represent the most abundant component of many eukaryotic genomes. Mobile elements are a driving force of eukaryotic genome evolution. McClintock's Ac, the autonomous element of the Ac/Ds system, together with hobo from Drosophila and Tam3 from snapdragon define an ancient and diverse DNA transposon superfamily named hAT. Other members of the hAT superfamily include the insect element Hermes and Tol2 from medaka. In recent years, genetic tools derived from the 'cut' and 'paste' Tol2 DNA transposon have been widely used for genomic manipulation in zebrafish, mammals and in cells in vitro.
  
  
  RESULTS
  We report the purification of a functional recombinant Tol2 protein from E.coli. We demonstrate here that following microinjection using a zebrafish embryo test system, purified Tol2 transposase protein readily catalyzes gene transfer in both somatic and germline tissues in vivo. We show that purified Tol2 transposase can promote both in vitro cutting and pasting in a defined system lacking other cellular factors. Notably, our analysis of Tol2 transposition in vitro reveals that the target site preference observed for Tol2 in complex host genomes is maintained using a simpler target plasmid test system, indicating that the primary sequence might encode intrinsic cues for transposon integration.
  
  
  CONCLUSIONS
  This active Tol2 protein is an important new tool for diverse applications including gene discovery and molecular medicine, as well as for the biochemical analysis of transposition and regulation of hAT transposon/genome interactions. The measurable but comparatively modest insertion site selection bias noted for Tol2 is largely determined by the primary sequence encoded in the target sequence as assessed through studying Tol2 protein-mediated transposition in a cell-free system.
authors.affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 1st St SW, 1342C Guggenheim, Rochester, MN 55905 USA ; Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94305 USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Ni
authors.affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455 USA ; University of Pennsylvania, 9 Penn Tower, 3400 Spruce ST, Philadelphia, PA 19104 USA.
authors.forename: Kirk J
authors.initials: KJ
authors.lastname: Wangensteen
authors.affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455 USA.
authors.forename: David
authors.initials: D
authors.lastname: Nelsen
authors.affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455 USA ; Department of Biology, Temple University, Philadelphia, PA 19122 USA.
authors.forename: Darius
authors.initials: D
authors.lastname: Balciunas
authors.affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 1st St SW, 1342C Guggenheim, Rochester, MN 55905 USA.
authors.forename: Kimberly J
authors.initials: KJ
authors.lastname: Skuster
authors.affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 1st St SW, 1342C Guggenheim, Rochester, MN 55905 USA.
authors.forename: Mark D
authors.initials: MD
authors.lastname: Urban
authors.affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 1st St SW, 1342C Guggenheim, Rochester, MN 55905 USA.
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Ekker
keywords: Recombinant transposase protein; Tol2 transposase; Transposition site preference; Zebrafish; hAT superfamily