title: Improved conditional expression systems resulting in physiological level of HNF4alpha expression confirm HNF4alpha induced apoptosis in the pancreatic beta-cell line INS-1.
abstract: BACKGROUND
  To analyze gene function in mammalian cells tetracycline inducible expression of a gene-of-interest at a specific genomic location (Flp-In T-REx) is most attractive. However, leakiness of basal transgene expression and artificially high expression level upon tetracycline addition may be disadvantageous.
  
  
  RESULTS
  To solve these problems, we developed two different approaches to improve our pancreatic beta-cell line INS-1 Flp-In T-REx expressing the tissue restricted transcription factor HNF4alpha under control of tetracycline. On the one hand we replaced the strong full length CMV promoter (CMV-Wt) with a weaker 5'-deleted CMV promoter fragment of 138 nucleotides in length (CMV-138). On the other hand we extended our INS-1 Flp-In T-REx cell lines with a Shield-1 dependent conditional control system of protein stability. Therefore, we fused HNF4alpha to the destabilization domain (DD) deduced from human FKBP12 protein. As a result in both approaches basal transgene expression level was markedly reduced, but HNF4alpha induction could still be maintained. Additionally, we could show that a low increase in HNF4alpha induces caspase activity indicating an apoptotic effect of HNF4alpha in these cells.
  
  
  CONCLUSIONS
  In the present study we considerably improved our INS-1 Flp-In T-REx cell lines conditionally expressing HNF4alpha to reduce leakiness and to optimize exogenous HNF4alpha protein expression to a physiological level. As an important result we could extend our previous results that HNF4alpha induces apoptosis in the pancreatic beta-cell line INS-1 with the new aspect that an expression level of the HNF4alpha transgene marginally exceeding the endogenous level is sufficient to trigger apoptosis.
authors.affiliation: Institut für Zellbiologie (Tumorforschung), Universitätsklinikum Essen, Universität, Duisburg-Essen, D-45122 Essen, Germany. sabine.senkel@uni-due.de
authors.forename: Sabine
authors.initials: S
authors.lastname: Senkel
authors.forename: Christoph
authors.initials: C
authors.lastname: Waldner
authors.forename: Gerhart U
authors.initials: GU
authors.lastname: Ryffel
authors.forename: Heike
authors.initials: H
authors.lastname: Thomas