title: Chimeric piggyBac transposases for genomic targeting in human cells.
abstract: Integrating vectors such as viruses and transposons insert transgenes semi-randomly and can potentially disrupt or deregulate genes. For these techniques to be of therapeutic value, a method for controlling the precise location of insertion is required. The piggyBac (PB) transposase is an efficient gene transfer vector active in a variety of cell types and proven to be amenable to modification. Here we present the design and validation of chimeric PB proteins fused to the Gal4 DNA binding domain with the ability to target transgenes to pre-determined sites. Upstream activating sequence (UAS) Gal4 recognition sites harbored on recipient plasmids were preferentially targeted by the chimeric Gal4-PB transposase in human cells. To analyze the ability of these PB fusion proteins to target chromosomal locations, UAS sites were randomly integrated throughout the genome using the Sleeping Beauty transposon. Both N- and C-terminal Gal4-PB fusion proteins but not native PB were capable of targeting transposition nearby these introduced sites. A genome-wide integration analysis revealed the ability of our fusion constructs to bias 24% of integrations near endogenous Gal4 recognition sequences. This work provides a powerful approach to enhance the properties of the PB system for applications such as genetic engineering and gene therapy.
authors.affiliation: Institute for Biogenesis Research, Department of Anatomy, Biochemistry, and Physiology, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI 96822, USA.
authors.forename: Jesse B
authors.initials: JB
authors.lastname: Owens
authors.forename: Johann
authors.initials: J
authors.lastname: Urschitz
authors.forename: Ilko
authors.initials: I
authors.lastname: Stoytchev
authors.forename: Nong C
authors.initials: NC
authors.lastname: Dang
authors.forename: Zoia
authors.initials: Z
authors.lastname: Stoytcheva
authors.forename: Mahdi
authors.initials: M
authors.lastname: Belcaid
authors.forename: Kommineni J
authors.initials: KJ
authors.lastname: Maragathavally
authors.forename: Craig J
authors.initials: CJ
authors.lastname: Coates
authors.forename: David J
authors.initials: DJ
authors.lastname: Segal
authors.forename: Stefan
authors.initials: S
authors.lastname: Moisyadi
mesh_terms: D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D018390:Gene Targeting; D016678:Genome; D057809:HEK293 Cells; D006801:Humans; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019895:Transposases / Q000235:genetics / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D019895:Transposases