"Was mache ich eigentlich in Schweden?"

haben sich bestimmt einige schon gefragt. Deshalb veröffentliche ich hier mal mein abstract. Wer nur Bahnhof versteht, dem kann ich nur sagen, dass weder ein dreibeiniges, 20äugiges Monster noch ein Klon meinerselbst dabei herauskommen wird.

Construction of an inducible expression system for hCYP2E1 in mammalian cells

The cytochrome P450 enzyme CYP2E1 is located in the ER membrane where it is involved in the metabolism of low molecular weight xenobiotica. The activity of CYP2E1 has been shown to generate high amounts of reactive oxygen species (ROS) and also to induce apoptosis. We want to establish a tightly regulated inducible expression system of this enzyme as a model system for how endogenously formed ROS can regulate the apoptotic machinery.
The expression system, which we will use, is based on the bacterial tetracycline-regulated repressor (TetR) together with a Flp recombinase-mediated DNA recombination into a single locus (a FRT locus). We have subcloned hCYP2E1 into a tetracycline regulated expression vector and transfected it into human embryonal kidney cells (293) containing a FRT site and TetR. Clones with hCYP2E1 integrated will be characterized in terms of regulation of CYP2E1 expression as well as enzyme activity. To assess the suitability of this 293 cell line as expression host we measure the cytochrome P450 reductase (CPR) activity. For an expression system with possibly higher CYP2E1 activity we will use a hepatoma cell line (C3A), which should have high CPR activity. These cells need to be manipulated to have a FRT site integrated as a single copy as well as to express the TetR. Accordingly, we have started to generate C3A cells to have a FRT site and TetR stably integrated in their genome.