Previous and current research

We study cell signalling in mammals by an approach that we call "in vivo biochemistry". To this end, we use state-of-the-art transgenic mouse technology and try to "watch" biochemical processes in real time in living cells, tissues and mice. Specifically, we are interested in the role of the signalling molecule cGMP in health and disease, with a current focus on cell growth and plasticity in the mammalian cardiovascular and nervous system. The projects involve analyses at the whole organism, cellular and molecular level.

Among the techniques used in the lab are:

General and more specialized methods of molecular biology, protein biochemistry and cell biology including:

Construction of plasmid vectors (including gene targeting vectors)
Expression of recombinant proteins in E. coli and in the baculovirus/insect cell system
Protein purification and biochemical analysis
Protein kinase assays
Generation of polyclonal antibodies
Mammalian cell culture
Embryonic stem cell culture and differentiation
Western blotting
Immunohistochemistry/Immunofluorescence of cells and tissue sections
Non-invasive monitoring of cell growth (live cell imaging, impedance-based systems)
Fluorescence imaging of signaling molecules in living cells (e.g. by FRET)
Gene and protein expression profiling (scRNAseq and proteomics)

Generation and phenotyping of transgenic cell and mouse models:

Gene targeting in embryonic stem cells
Time- and tissue-specific somatic mutagenesis using the Cre/lox recombination system
Tamoxifen-inducible CreER recombinases
Isolation, culture, and functional analysis of primary cells from transgenic mice
Biochemical characterization of signalling pathways in transgenic cells and mice
Mouse models of atherosclerosis and restenosis
Behavioural testing (e.g. for learning and motor coordination)
Telemetric monitoring of physiological parameters (heart rate, blood pressure, etc.)
Non-invasive anatomical and functional imaging of mice by MR, CT and PET (in cooperation with B. Pichler)

Conditional gene targeting

In order to understand the role of a given gene product in a given cell type at a given developmental stage, genetic techniques are being developed that allow for the introduction of defined mutations into the mouse genome at will, in a specific cell type and at a chosen time. Most current conditional gene targeting systems are based on the use of the site-specific recombinase Cre which catalyzes recombination between two 34 bp loxP recognition sites.

The basic strategy for Cre/loxP-directed gene knockout experiments is to flank (‘flox’) an essential exon of the gene of interest with two loxP sites (by homologous recombination in ES cells), and then to ‘deliver’ Cre to excise the intervening DNA including the exon from the chromosome, thus generating a null allele in all cells where Cre is active. ‘Delivery’ of Cre can be achieved by crossing mice carrying the ‘floxed’ target gene with transgenic Cre-expressing mice. Clearly, key to successful conditional gene targeting is the availability of Cre transgenic mouse strains in which Cre activity is tightly controlled in space and time.

To this end, we have developed ligand-dependent chimeric Cre recombinases, so-called CreER recombinases. They consist of Cre fused to mutated hormone binding domains of the estrogen receptor. The CreER recombinases are inactive, but can be activated by the synthetic estrogen receptor ligand tamoxifen, therefore allowing for external temporal control of Cre activity. Indeed, by combining tissue-specific expression of a CreER recombinase with its tamoxifen-dependent activity, the excision of ‘floxed’ chromosomal DNA can be controlled both spatially and temporally by administration of tamoxifen to the mouse. Our current efforts are primarily directed towards further refining ligand-activated site-specific recombination in mice. Specifically, we try...

... to increase the efficiency of the system in mice by using novel ‘high-sensitivity’ CreER-like recombinases,

... to develop CreER-based methods to genetically label wild-type and knockout cells and to follow their fate during embryonic development or during disease progression in the adult animal (Cre/lox-assisted cell fate mapping)

... to use spatio-temporally controlled somatic mutagenesis to study the role of selected genes in the cardiovascular and nervous system.

(a) Tissue-specific gene inactivation is based on excision of a loxP(triangle)-flanked exon (E) in Cre (C)-expressing cells (shaded oval).
(b) Temporal control over recombination can be obtained by using a ligand-dependent Cre recombinase (LC) that is inactive in the absence (boxed LC) and active in the presence (circled LC*) of a synthetic ligand (*). Spatio-temporally controlled somatic mutagenesis can be achieved by tissue-specific expression of a ligand-dependent recombinase.

cGMP signalling & cell plasticity

The second messenger cGMP mediates many beneficial and perhaps also detrimental effects of the endogenous signalling molecules nitric oxide, carbon monoxide and natriuretic peptides. Drugs that modulate the intracellular cGMP level are used to treat various human diseases, for instance, organic nitrates (e.g. glyceroltrinitrate) for coronary heart disease/angina pectoris and phosphodiesterase inhibitors (e.g. sildenafil [Viagra®]) for erectile dysfunction and pulmonary hypertension. A downstream effector of cGMP is the cGMP-dependent protein kinase type I (cGKI). The role of cGKI in mediating short-term effects of cGMP, like vasorelaxation and modulation of platelet aggregation, has been intensively studied. However, the long-term effects of cGMP-cGKI signalling on the cell phenotype and associated (patho-)physiological processes are not clear. We have established conditional mouse mutants with defined cGKI mutations in selected cell types. The analysis of these mice indicates that cGKI is an important mediator of cGMP in the cardiovascular and nervous system, opening new therapeutic options for major human diseases. Of particular interest, the cGMP/cGKI system appears to be critically involved in the regulation of cell growth, survival and plasticity, ranging from the change of smooth muscle cell phenotype during atherosclerosis to axonal pathfinding and the modulation of neuronal activity during learning and nociception.

Future projects

A major aim of our future projects is to improve our understanding of the in vivo biochemistry of cGMP signalling under normal and pathological conditions. We will use conditional genetic approaches combined with live cell imaging to further dissect the molecular mechanisms that regulate cell plasticity during vascular remodeling and tumor development. An important question is how the cGMP-cGKI system regulates proliferation, apoptosis, and differentiation of cells. Based on our recent discovery of mechanosensitive cGMP signaling (mechano-cGMP), i.e. cGMP signals are also modulated by mechanical force acting on cells, we are also interested to dissect the mechanisms of this novel signaling mode as well as its (patho-)physiological and therapeutic relevance. We will also further develop the conditional recombination technology, for instance, to follow the fate of selected cells during disease processes (cell fate mapping).

Goals

To study biochemistry and cell biology in a living mammalian organism to gain new insights into complex biological systems.

In particular, to understand the role of cellular plasticity in physiological and pathological processes such as learning, ageing, atherosclerosis, and cancer.