title: Tn5 as an insect gene vector.
abstract: The purpose of this study was to explore alternatives to insect-derived transposable elements as insect gene vectors with the intention of improving existing insect transgenesis methods. The mobility properties of the bacterial transposon, Tn5, were tested in mosquitoes using a transient transposable element mobility assay and by attempting to create transgenic insects. Tn5 synaptic complexes were assembled in vitro in the absence of Mg(2+) and co-injected with a target plasmid into developing yellow fever mosquito, Aedes aegypti, embryos. Target plasmids recovered from embryos a day later were screened for the presence of Tn5. Recombinants (transposition events) were found at a frequency of 1.2 x 10(-3). Some transposition events did not appear to be associated with canonical 9 bp direct duplications at the site of insertion and also were associated with either deletions or rearrangements. A Tn5 element containing the brain-specific transgene, 3 x P3DsRed, was assembled into synaptic complexes in vitro and injected into pre-blastoderm embryos of Ae. aegypti. Of the approximately 900 embryos surviving injection and developing into adults, two produced transgenic progeny. Both transgenic events involved the co-integrations of approximately five elements resulting in nested and tandem arrayed Tn5::3 x P3DsRed elements. This study extends the known host range of Tn5 to insects and makes available to insect biologists and others another eukaryotic genome-manipulation tool. The hyperactivity of synaptic complexes may be responsible for the unusual clustering of elements and managing this aspect of the element's behavior will be important in future applications of this technology to insects.
authors.affiliation: Center for Biosystems Research, University of Maryland Biotechnology Institute, Plant Sciences Building/Room 5115, College Park, MD 20742-4450, USA.
authors.forename: Kathryn H
authors.initials: KH
authors.lastname: Rowan
authors.forename: Jamison
authors.initials: J
authors.lastname: Orsetti
authors.forename: Peter W
authors.initials: PW
authors.lastname: Atkinson
authors.forename: David A
authors.initials: DA
authors.lastname: O'Brochta
mesh_terms: D000330:Aedes / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001756:Blastoderm / Q000502:physiology; D017931:DNA Primers; D004625:Embryo, Nonmammalian / Q000502:physiology; D005822:Genetic Vectors; D007313:Insecta / Q000201:enzymology / Q000235:genetics*; D009024:Morphogenesis; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D014162:Transfection / Q000379:methods; D019895:Transposases / Q000235:genetics*
chemical_list: D017931:DNA Primers; C473503:Tn5 transposase; D019895:Transposases