title: An in vivo transfection system for inducible gene expression and gene silencing in murine hepatocytes.
abstract: BACKGROUND
  Hydrodynamic tail vein injection (HTVI) of transposon-based integration vectors is an established system for stably transfecting mouse hepatocytes in vivo that has been successfully employed to study key questions in liver biology and cancer. Refining the vectors for transposon-mediated hepatocyte transfection will further expand the range of applications of this technique in liver research. In the present study, we report an advanced transposon-based system for manipulating gene expression in hepatocytes in vivo.
  
  
  METHODS
  Transposon-based vector constructs were generated to enable the constitutive expression of inducible Cre recombinase (CreER) together with tetracycline-inducible transgene or miR-small hairpin RNA (shRNA) expression (Tet-ON system). Transposon and transposase expression vectors were co-injected into R26R-mTmG reporter mice by HTVI. Cre-mediated gene recombination was induced by tamoxifen, followed by the administration of doxycycline to drive tetracycline-inducible gene or shRNA expression. Expression was visualized by immunofluorescence staining in livers of injected mice.
  
  
  RESULTS
  After HTVI, Cre recombination by tamoxifen led to the expression of membrane-bound green fluorescent protein in transfected hepatocytes. Activation of inducible gene or shRNA expression was detected by immunostaining in up to one-third of transfected hepatocytes, with an efficiency dependent on the promoter driving the Tet-ON system.
  
  
  CONCLUSIONS
  Our vector system combines Cre-lox mediated gene mutation with inducible gene expression or gene knockdown, respectively. It provides the opportunity for rapid and specific modification of hepatocyte gene expression and can be a useful tool for genetic screening approaches and analysis of target genes specifically in genetically engineered mouse models.
authors.affiliation: Department of Medicine II, Klinikum rechts der Isar, Technische Universität München, Munich, Germany.
authors.forename: Eric K
authors.initials: EK
authors.lastname: Hubner
authors.affiliation: Department of Medicine II, Klinikum rechts der Isar, Technische Universität München, Munich, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Lechler
authors.affiliation: Department of Medicine II, Klinikum rechts der Isar, Technische Universität München, Munich, Germany.
authors.forename: Birgit
authors.initials: B
authors.lastname: Kohnke-Ertel
authors.affiliation: Department of Pediatrics and Genetics, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: Anne-Flore
authors.initials: AF
authors.lastname: Zmoos
authors.affiliation: Department of Pediatrics and Genetics, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: Julien
authors.initials: J
authors.lastname: Sage
authors.affiliation: Department of Medicine II, Klinikum rechts der Isar, Technische Universität München, Munich, Germany.
authors.forename: Roland M
authors.initials: RM
authors.lastname: Schmid
authors.affiliation: Department of Medicine II, Klinikum rechts der Isar, Technische Universität München, Munich, Germany.
authors.forename: Ursula
authors.initials: U
authors.lastname: Ehmer
mesh_terms: D000818:Animals; D004251:DNA Transposable Elements; D004318:Doxycycline / Q000494:pharmacology; D015870:Gene Expression; D005786:Gene Expression Regulation / Q000187:drug effects; D055786:Gene Knockout Techniques; D023061:Gene Order; D020868:Gene Silencing; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000008:administration & dosage / Q000235:genetics; D022781:Hepatocytes / Q000378:metabolism*; D059765:Homologous Recombination; D051379:Mice; D011401:Promoter Regions, Genetic; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection / Q000379:methods; D019076:Transgenes
chemical_list: D004251:DNA Transposable Elements; D034741:RNA, Small Interfering; D004318:Doxycycline
keywords: HTVI; Tet-ON system; hydrodynamic tail vein injection; inducible gene expression; sleeping beauty transposon