1 Det Sundhedsvidenskabelige Fakultet, SDU2 Moleculær Oncology Group / Mollenhauer group, Department of Molecular Medicine, Det Sundhedsvidenskabelige Fakultet, SDU3 Moleculær Oncology Group / Mollenhauer group, Department of Molecular Medicine, Det Sundhedsvidenskabelige Fakultet, SDU
Optimization of novel vector systems for functional genomics in cancer research Steffen Schmidt1*, Stephanie Blaich2, Rainer Wittig3, Stefan Lyer4, Caroline End2, Melanie Hudler2, Lukasz Kacprzyk2, Angela Riedel1,2, Helle Christiansen1, Jan Mollenhauer1,2 1 Molekylær Onkologi, Medicinsk Bioteknologisk Center, Institut for Medicinsk Biologi, Syddansk Universitet, JB Winsløws Vej 25, 5000 Odense, Danmark 2 Afdeling for Molekylær Genomanalyse, DKFZ, Im Neuenheimer Feld 280, 69120 Heidelberg, Tyskland 3 Institut for Laser Teknologi, Helmholtzstrasse 12, 89081 Ulm Universitet, Tyskland 4 Institut for Lokal Tumor Terapi, Universitet Hospital, Waldstraße1, 91054 Erlangen, Tyskland * Præsenterende forfatter Large datasets about differentially expressed genes in cancer tissue have been recovered by expression profiling using microarray technologies. To study the effects of these genes in cancer cells will lead to an improved understanding of the molecular mechanisms underlying cancer and may result in the identification of novel druggable targets for cancer treatment. We established a novel rapid technique to generate stable cell lines with inducible overexpression of genes. This enables for functional analyses of the genes’ effects on cancer-relevant functions at a considerable scale. First, a vector with a sequence for site-specific recombination is inserted into the genome of the cancer cell line to generate an acceptor cell line. In a second step, the expression vector containing the gene of interest under the control of a tetracycline inducible promoter can easily be inserted into the acceptor cell line genome by co-transfection with a recombinase-coding plasmid. While this represents and extremely powerful technique, we found that silencing efficacy of the tetracycline-inducible system varies in different cancer backgrounds. Using the same vector systems, virtually no leakiness is observed in A375 melanoma cells, while AGS gastric carcinoma cells seem to be resistant to Tet-repressor-mediated silencing. We developed a general qRT-PCR approach utilizing a tag that is common for all genes in our expression vector, which allows for directly measuring and comparing leakiness and inducible expression in different cancer cell lines. As the next step, we created siRNAs against the common tag, which were able to potently counteract leakiness of the Tet-inducible system in AGS cells. These data indicate that inclusion of RNAi-mediated silencing in inducible expression systems could potentially be able to overcome cell line-specific differences in the performance of inducible expression systems. This would represent a valuable progress towards the systematic functional analysis of novel cancer genes.