Dataset Information

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.

Findings

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.

Conclusion

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.

SUBMITTER: Senkel S

PROVIDER: S-EPMC2768738 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Publications

Improved conditional expression systems resulting in physiological level of HNF4alpha expression confirm HNF4alpha induced apoptosis in the pancreatic beta-cell line INS-1.

Senkel Sabine S Waldner Christoph C Ryffel Gerhart U GU Thomas Heike H

BMC research notes 20091017

<h4>Background</h4>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.<h4>Findings</h4>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 tran ...[more]

PMID: 19835622

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Project description:UnlabelledIntroductionNon-transformed mammary epithelial cell lines such as MCF-10A recapitulate epithelial morphogenesis in three-dimensional (3D) tissue culture by forming acinar structures. They represent an important tool to characterize the biological properties of oncogenes and to model early carcinogenic events. So far, however, these approaches were restricted to cells with constitutive oncogene expression prior to the set-up of 3D cultures. Although very informative, this experimental setting has precluded the analysis of effects caused by sudden oncoprotein expression or withdrawal in established epithelial cultures. Here, we report the establishment and use of a stable MCF-10A cell line (MCF-10Atet) fitted with a novel and improved doxycycline (dox)-regulated expression system allowing the conditional expression of any transgene.MethodsMCF-10Atet cells were generated by stable transfection with pWHE644, a vector expressing a second generation tetracycline-regulated transactivator and a novel transcriptional silencer. In order to test the properties of this new repressor/activator switch, MCF-10Atet cells were transfected with a second plasmid, pTET-HABRAF-IRES-GFP, which responds to dox treatment with the production of a bi-cistronic transcript encoding hemagglutinin-tagged B-Raf and green fluorescent protein (GFP). This improved conditional expression system was then characterized in detail in terms of its response to various dox concentrations and exposure times. The plasticity of the phenotype provoked by oncogenic B-RafV600E in MCF-10Atet cells was analyzed in 3D cultures by dox exposure and subsequent wash-out.ResultsMCF-10Atet cells represent a tightly controlled, conditional gene expression system. Using B-RafV600E as a model oncoprotein, we show that its sudden expression in established 3D cultures results in the loss of acinar organization, the induction of an invasive phenotype and hallmarks of epithelial-to-mesenchymal transition (EMT). Importantly, we show for the first time that this severe transformed phenotype can be reversed by dox wash-out and concomitant termination of oncogene expression.ConclusionsTaken together, we have generated a stable MCF-10A subline allowing tight dox-controlled and reversible expression of any transgene without the need to modify its product by introducing artificial dimerization or ligand-binding domains. This system will be very valuable to address phenomena such as EMT, oncogene addiction, oncogene-induced senescence and drug resistance.

Dataset Information

Improved conditional expression systems resulting in physiological level of HNF4alpha expression confirm HNF4alpha induced apoptosis in the pancreatic beta-cell line INS-1.

Background

Findings

Conclusion

Publications

Improved conditional expression systems resulting in physiological level of HNF4alpha expression confirm HNF4alpha induced apoptosis in the pancreatic beta-cell line INS-1.

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