Improved conditional expression systems resulting in physiological level of HNF4α expression confirm HNF4α induced apoptosis in the pancreatic β-cell line INS-1

Abstract

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 β-cell line INS-1 Flp-In T-REx™ expressing the tissue restricted transcription factor HNF4α 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 HNF4α to the destabilization domain (DD) deduced from human FKBP12 protein. As a result in both approaches basal transgene expression level was markedly reduced, but HNF4α induction could still be maintained. Additionally, we could show that a low increase in HNF4α induces caspase activity indicating an apoptotic effect of HNF4α in these cells. Conclusion In the present study we considerably improved our INS-1 Flp-In T-REx™ cell lines conditionally expressing HNF4α to reduce leakiness and to optimize exogenous HNF4α protein expression to a physiological level. As an important result we could extend our previous results that HNF4α induces apoptosis in the pancreatic β-cell line INS-1 with the new aspect that an expression level of the HNF4α transgene marginally exceeding the endogenous level is sufficient to trigger apoptosis.