title: Cis-acting gene regulatory activities in the terminal regions of sleeping beauty DNA transposon-based vectors.
abstract: Sleeping Beauty (SB) DNA transposon-based vectors belong to a growing family of nonviral integrating vectors that represent attractive alternatives to conventional virus-based integrating gene vehicles. Because of concerns related to mutagenesis and/or activation of cellular genes by integrating vectors, much attention has been paid to integration site preferences and the ability of vectors to influence expression of neighboring genes. Here, we test the hypothesis that terminal repeats of transposons carry cis-acting regulatory sequences. In transient gene expression studies, we demonstrate that the inverted repeats of SB direct gene expression in HeLa cells to levels that are 3-fold higher than in promoter-deficient controls. Inverted repeats pointing toward the transposon center consistently facilitate the highest levels of activity in a number of cell lines. We show that transposon sequences flanking the inverted repeats of SB are required for positive effects on gene expression and, moreover, that these regions contain both stimulatory and inhibitory cis-acting elements. In the context of an integrated SB vector the regulatory activities of the transposon termini are sufficient to drive expression of selectable marker genes carried by the transposon, indicating that opposing transcriptional activities originating from the transposon termini may influence expression of its genetic cargo. Finally, detection of regulatory properties of the terminal repeats of the active Tc3 element from Caenorhabditis elegans leads to the suggestion that transcriptional activities of the inverted repeats are conserved among Tc1/mariner transposons in nature. Our data suggest that SB-based gene vectors may carry ancient properties of self-regulation with potential relevance for SB-directed therapeutic gene transfer.
authors.affiliation: Department of Human Genetics, University of Aarhus, DK-8000 Aarhus, Denmark.
authors.forename: Brian
authors.initials: B
authors.lastname: Moldt
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Yant
authors.forename: Peter Refsing
authors.initials: PR
authors.lastname: Andersen
authors.forename: Mark A
authors.initials: MA
authors.lastname: Kay
authors.forename: Jacob Giehm
authors.initials: JG
authors.lastname: Mikkelsen
mesh_terms: D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000235:genetics; D004251:DNA Transposable Elements / Q000235:genetics*; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D020079:Terminal Repeat Sequences
chemical_list: D004251:DNA Transposable Elements; D008156:Luciferases