title: Analysis of site-specific transgene integration following cotransduction with recombinant adeno-associated virus and a rep encodingplasmid.
abstract: BACKGROUND
  Recombinant adeno-associated virus (rAAV) has many advantages for gene therapeutic applications in comparison with other vector systems. One of the most promising features is the ability of wild-type (wt) AAV to integrate site-specifically into human chromosome 19. However, this feature is lost in rAAV vectors due to the removal of the rep-coding sequences.
  
  
  METHODS
  HeLa cells were transfected with a rep expression plasmid, infected by rAAV and grown with or without selection pressure. Single cell clones were generated and genomic DNA was analyzed for site-specific integration by Southern blotting analysis and fluorescence in situ hybridization (FISH).
  
  
  RESULTS
  Transfection of HeLa cells with a rep expression plasmid followed by transduction with a rAAV vector resulted in site-specific integration of the transgene at AAVS1 on human chromosome 19 in 7 of 10 cell clones analyzed. In marked contrast, transduction of cells with rAAV alone did not result in any site-specific integration of the transgene.
  
  
  CONCLUSIONS
  The high frequency with which the site-specific integration took place in the presence of Rep protein is comparable with the results observed with wtAAV. These results offer opportunities for the development of specifically integrating rAAV vectors.
authors.affiliation: Gene Center of the Ludwig-Maximilians-University Munich, Feodor-Lynen-Str. 25, 81377 Munich, Germany.
authors.forename: Nadja A
authors.initials: NA
authors.lastname: Huttner
authors.forename: Anne
authors.initials: A
authors.lastname: Girod
authors.forename: Susanne
authors.initials: S
authors.lastname: Schnittger
authors.forename: Claudia
authors.initials: C
authors.lastname: Schoch
authors.forename: Michael
authors.initials: M
authors.lastname: Hallek
authors.forename: Hildegard
authors.initials: H
authors.lastname: Büning
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D002888:Chromosomes, Human, Pair 19 / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D010957:Plasmids / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic; D019076:Transgenes; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C068734:rep proteins, Adeno-associated virus 2