Technology Development
The Technology development team works on the development and implementation of new stem cell and mutagenesis tools. We focus on the use and development of zinc-finger and TAL nucleases for the one-step generation of targeted mutants in fertilised oocytes (Figure), the development of vector-free iPS cells and the control of signaling proteins by fusion with the tamoxifen regulated ERT2 (estrogen receptor) domain.
Group and Teamleaders, from left: Wolfgang Wurst, Jens Hansen, Ralf Kühn, Joel Schick
Absent: Thomas Floss
Most important publications:
Meyer M, de Angelis MH, Wurst W, Kühn R. (2010). Gene targeting by homologous recombination in mouse zygotes mediated by zinc-finger nucleases. Proc Natl Acad Sci USA, 107, 15022-15026.
Steuber-Buchberger, P., Wurst, W. and Kühn, R. (2008). Simultaneous Cre-mediated conditional knockdown of two genes in mice. Genesis 46, 144 -151.
Delic, S., Streif, S. Deussing, J.M., Weber, P., Ueffing, M., Hölter, S.M., Wurst, W. and Kühn, R. (2008). Genetic Mouse Models for Behavioral Analysis through Transgenic RNAi Technology. Genes Brain Behav.7, 821 – 830.
Chu, Y., Senghaas, N., Köster, R.W., Wurst, W. and Kühn, R. (2008). Novel Caspase-Suicide Proteins for Tamoxifen-Inducible Apoptosis. Genesis, 46, 530- 536.
Hitz, C. Wurst, W. and Kühn, R. (2007). Conditional brain-specific knockdown of MAPK using Cre/loxP regulated RNA interference. Nucleic Acids Res. 35, e90
Large-scale Targeting
The Large-scale targeting group (J. Schick) develops technology for the rapid assembly of targeting constructs. Until recently, targeting vectors were generated externally using recombineering or assembled by standard step-by-step cloning in E. coli. A new and efficient in-house method of targeting vector construction expedites our efforts to modify the ES/iPS cell genome. To this end, we developed a new yeast cloning system that for the first time enables rapid targeting vector engineering (within one week) and ES mutagenesis for internal projects and future large-scale functional projects. Additional research projects in the group aim to use novel genetic tools to promote differentiation of therapeutically relevant cell types in the CNS from iPS/ES cells, as well as unravel potential mechanisms of de- and transdifferentiation in somatic cells. This projects are divided into the (i) characterization and genetic modification of iPS cell lines, (ii) use of cell engineering in in vitro differentiation, and (iii) the use of automation and parallel techniques in drug discovery and neuroscience (i.e., chemical and genetic (transposon) screens).
- Overlapping DNA fragments are easily assembled in yeast to make targeting vectors.
- An overview of our research pipeline of in vitro neurodegenerative disease models. Targeting vectors made using the yeast system are used to establish marker ES/iPS cell lines, which are targeted in a second round to overexpress transcription factors important for differentiation. Finally, human gene variants found in neurodegenerative patients are engineered into the cells.