title: RNA interference is responsible for reduction of transgene expression after Sleeping Beauty transposase mediated somatic integration.
abstract: BACKGROUND
  Integrating non-viral vectors based on transposable elements are widely used for genetically engineering mammalian cells in functional genomics and therapeutic gene transfer. For the Sleeping Beauty (SB) transposase system it was demonstrated that convergent transcription driven by the SB transposase inverted repeats (IRs) in eukaryotic cells occurs after somatic integration. This could lead to formation of double-stranded RNAs potentially presenting targets for the RNA interference (RNAi) machinery and subsequently resulting into silencing of the transgene. Therefore, we aimed at investigating transgene expression upon transposition under RNA interference knockdown conditions.
  
  
  RESULTS
  To establish RNAi knockdown cell lines we took advantage of the P19 protein, which is derived from the tomato bushy stunt virus. P19 binds and inhibits 21 nucleotides long, small-interfering RNAs and was shown to sufficiently suppress RNAi. We found that transgene expression upon SB mediated transposition was enhanced, resulting into a 3.2-fold increased amount of colony forming units (CFU) after transposition. In contrast, if the transgene cassette is insulated from the influence of chromosomal position effects by the chicken-derived cHS4 insulating sequences or when applying the Forg Prince transposon system, that displays only negligible transcriptional activity, similar numbers of CFUs were obtained.
  
  
  CONCLUSIONS
  In summary, we provide evidence for the first time that after somatic integration transposon derived transgene expression is regulated by the endogenous RNAi machinery. In the future this finding will help to further improve the molecular design of the SB transposase vector system.
authors.affiliation: Max von Pettenkofer-Institute, Department of Virology, Ludwig-Maximilians-University Munich, Munich, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Rauschhuber
authors.forename: Anja
authors.initials: A
authors.lastname: Ehrhardt
mesh_terms: D002460:Cell Line; D004251:DNA Transposable Elements / Q000235:genetics; D015870:Gene Expression; D005786:Gene Expression Regulation; D023061:Gene Order; D020868:Gene Silencing; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D034622:RNA Interference; D019076:Transgenes; D019895:Transposases / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism
chemical_list: D004251:DNA Transposable Elements; C493885:P19 protein, tomato bushy stunt virus; D014764:Viral Proteins; D019895:Transposases