title: DNA nuclear targeting sequences for non-viral gene delivery.
abstract: OBJECTIVE
  To evaluate if introduction of DNA nuclear Targeting Sequences (DTS; i.e. recognition sequences for endogenous DNA-binding proteins) in plasmid DNA (pDNA) leads to increased transfection efficiency of non-viral gene delivery by virtue of enhanced nuclear import of the pDNA.
  
  
  METHODS
  A set of DTS was identified and cloned into EGFP-reporter plasmids controlled by the CMV-promoter. These pDNA constructs were delivered into A431 and HeLa cells using standard electroporation, pEI-based polyfection or lipofection methods. The amount of pDNA delivered into the nucleus was determined by qPCR; transfection efficiency was determined by flow cytometry.
  
  
  RESULTS
  Neither of these DTS increased transgene expression. We varied several parameters (mitotic activity, applied dose and delivery strategy), but without effect. Although upregulated transgene expression was observed after stimulation with TNF-α, this effect could be ascribed to non-specific upregulation of transcription rather than enhanced nuclear import. Nuclear copy numbers of plasmids containing or lacking a DTS did not differ significantly after lipofectamine-based transfection in dividing and non-dividing cells.
  
  
  CONCLUSIONS
  No beneficial effects of DTS on gene expression or nuclear uptake were observed in this study.
authors.affiliation: Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA, Utrecht, The Netherlands.
authors.forename: Ethlinn V B
authors.initials: EV
authors.lastname: van Gaal
authors.forename: Ronald S
authors.initials: RS
authors.lastname: Oosting
authors.forename: Roel
authors.initials: R
authors.lastname: van Eijk
authors.forename: Marta
authors.initials: M
authors.lastname: Bakowska
authors.forename: Dries
authors.initials: D
authors.lastname: Feyen
authors.forename: Robbert Jan
authors.initials: RJ
authors.lastname: Kok
authors.forename: Wim E
authors.initials: WE
authors.lastname: Hennink
authors.forename: Daan J A
authors.initials: DJ
authors.lastname: Crommelin
authors.forename: Enrico
authors.initials: E
authors.lastname: Mastrobattista
mesh_terms: D001483:Base Sequence; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000235:genetics; D018274:Electroporation; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction