Published online 1 November 2004
Nucleic Acids Research, Vol. 32 No. 19 © Oxford University Press 2004; all rights reserved
Pattern of genes influenced by conditional expression of the transcription factors HNF6, HNF4
and HNF1ß in a pancreatic ß-cell line
Institut für Zellbiologie (Tumorforschung), Universitätsklinikum Essen, D-45122 Essen, Germany
* To whom correspondence should be addressed. Tel: +49 201 723 3110; Fax: +49 201 723 5905; Email: gerhart.ryffel{at}uni-essen.de
Received July 26, 2004; Revised September 22, 2004; Accepted October 6, 2004
Using the rat insulinoma cell line INS-1 we generated ß-cell clones that are most efficient for gene transfer, as they contain an FRT site for Flp recombinase-mediated, site-directed integration of a single copy transgene. Therefore, the gene-of-interest can be introduced by DNA transfection without the need to select individual cell clones. Additionally, the clones contain the tetracycline repressor allowing tetracycline induction of the transgene. By oligonucleotide microarray we define the ß-cell specific phenotype of the Flp-In T-REx cell clones. Using a clone expressing the HNF6, HNF4
and HNF1ß transcription factors at a limited level, we introduced the expression vectors encoding these factors. We show efficient tetracycline induction of these transcription factors by western blots and immunocytochemistry. Microarrays reveal that these three factors affect a similar number of genes with only few genes regulated in common. Statistical analysis reveals that the three transcription factors affect genes categorized to different biological processes. Furthermore, we document the usefulness of these Flp-In T-REx cells for the functional analysis of mutated HNF1ß transcription factors found in human MODY5 patients. We show that the expression of the mutant P328L329del and A263insGG affects only very few transcripts and these are predominantly distinct from those induced by wild-type HNF1ß.
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