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

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.