Improved conditional expression systems resulting in physiological level of HNF4 expression confirm HNF4 induced apoptosis in the pancreatic -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. Findings. To solve these problems, we developed two different approaches to improve our pancreatic -cell line INS-1 Flp-In T-REx expressing the tissue restricted transcription factor HNF4 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 HNF4 to the destabilization domain (DD) deduced from human FKBP12 protein. As a result in both approaches basal transgene expression level was markedly reduced, but HNF4 induction could still be maintained. Additionally, we could show that a low increase in HNF4 induces caspase activity indicating an apoptotic effect of HNF4 in these cells. Conclusion. In the present study we considerably improved our INS-1 Flp-In T-REx cell lines conditionally expressing HNF4 to reduce leakiness and to optimize exogenous HNF4 protein expression to a physiological level. As an important result we could extend our previous results that HNF4 induces apoptosis in the pancreatic -cell line INS-1 with the new aspect that an expression level of the HNF4 transgene marginally exceeding the endogenous level is sufficient to trigger apoptosis. © 2009 Thomas et al.