Precise regulation of transgene expression level and control of cell physiology

The elucidation of biological processes is inevitably linked with cells. To study these processes in basic research and in applied disciplines, cells usually are genetically manipulated. For the generation of biological meaningful data, a precise control of the transgene expression level is highly desirable. As cells are complex biological systems, the establishment of cell lines with such a predictable and/or controllable expression pattern is challenging. We have developed several techniques to achieve this goal with reasonable efforts. (1) Site-specific integration [molecular cut and paste mechanism (MCaP)] allows the generation of cell lines stably expressing the gene of interest within 4 weeks. In addition, these cell lines are characterised by a high and stable expression of the transgene. (2) The modulation of the transgene level can be easily achieved through transcriptional regulation. We found that the setup of the expression cassettes dramatically influences their expression pattern. (3) Cells reflecting in vivo properties are of high interest for life sciences. We have developed a system for cell expansion, which retains relevant cellular properties. The system is based on transcriptional control of expansion genes. The employed switch is mediated through the tetracycline system and allows strict and reversible control of cell proliferation.