A BSTRACTS 10.2217/17460751.2.6.3 © 2007 Future Medicine Ltd ISSN 1746-0751 Regen. Med. (2007) 2(6, Suppl.), 487–522 3 4th International Meeting Stem Cell Network North Rhine Westphalia Modulating stem cell properties by transducible Oct4 and Sox2 transcription factors Manal Bosnali † and Frank Edenhofer 1 † Corresponding author: Institute for Reconstructive Neurobiology, University of Bonn, Sigmund-Freud-Straße 25, 53127, Bonn, NRW, Germany; Tel.:: +49 228 6885 528; Email: mbosnali@uni-bonn.de 1 Institute for Reconstructive Neurobiology, Bonn, Germany Keywords: Oct4, pluripotency, Sox2 Stem cells have the unique capacities to self-renew and give rise to differentiated cell types. Gaining control over the mecha- nisms of stemness may provide a means to promote unlimited proliferation and/or dedifferentiation. Oct4 and Sox2 have been identified as key regulators of pluripotency. Together with the homeodomain protein Nanog these factors shape a core tran- scriptional network that controls two types of target genes with opposing functions: pluripotency genes and differentiation genes. In order to analyze this stemness transcriptional network we aimed to generate cell permeable versions of these factors, which can be used for direct cellular manipulation employing cell-permeant biologically active proteins. This method enables appli- cation of transcription factors in a controlled manner without genetic modification of the target cells. For that we tagged proteins with a protein transduction domain (TAT-sequence), which enables them to pass the cell mem- brane. In this study we engineered different versions of the transducible factors to identify the optimal candidates for recombi- nant expression and purification of the proteins. The purified recombinant proteins are able to bind specifically their corresponding DNA target sequences indicating functionality of the proteins. The path of the recombinant proteins into cul- tured cells was visualized by rhodamine labeling. Addition of the TAT-sequence to the protein enhanced its cellular uptake. We observed that transduction of each factor Oct4 and Sox2 resulted in improved stem cell properties as judged by the morphology of ES-cell colonies. Self renewal & pluripotency 1.