Investigating expression, function and biocompatibility of the genetically encoded calcium indicator TN-XXL in transgenic mice

Monitoring cellular calcium levels by fluorescent probes has become an important method to visualize cellular signaling processes. The development of genetically encoded calcium indicators (GECIs) strongly contributed to the application of calcium imaging which in contrast to synthetic calcium dyes enables cell-type specific labeling, long-term expression and therefore chronic imaging approaches. Whilst GECI engineering and optimization mainly focused on improving signal strength or calcium affinities and kinetics, little is known about the magnitude of interference of GECIs affecting cellular calcium homeostasis in host organisms. Various previous attempts to genetically express GECIs using calmodulin-based indicators frequently resulted in pathological changes or decreased function of the GECI in the host organism. In contrast to calmodulin-based GECIs, calcium imaging studies in the nervous system of flies and mice expressing troponin C-based GECIs did not display detrimental changes. In this work, TN-XXL performance was confirmed by transgenic TN-XXL expression in a Thy1.2 driven mouse line which enabled chronic in vivo calcium imaging of sensory evoked calcium transients of layer 2/3 neurons of the visual cortex during monocular deprivation over weeks. In addition, a detailed analysis focused on TN-XXL expression, function, viability and biocompatibility in mice ubiquitously expressing TN-XXL under the β-actin promoter. TN-XXL expression was determined in almost all tissues examined and functionality was validated in a variety of cultured cell-types. In addition the applicability of ratiometric indicators for calcium imaging of dynamic and complex tissues was evaluated via the use of explants from developing and adult cardiac tissues. Moreover, the tolerance of long-term expressed TN-XXL was verified on cellular level and along with a genome wide transcriptional analysis and behavioral experiments TN-XXL biocompatibility was investigated with only minor effects on the host genome and behavior. Together this study not only establishes the first detailed combined observation of function and biocompatibility of a GECI ubiquitously expressed in a mouse model but also has set the foundation criteria necessary for the evaluation of transgenic mouse lines expressing GECIs. Transgenic TN-XXL expression in this β-actin mouse line further offers ease of use for calcium signaling studies in a variety of cell-types and tissues of pharmaceutical relevance. Moreover, the facilitated cell line production and the possibility to establish mouse line crossings with existing disease models can open new grounds for investigations of calcium related diseases in vitro and in vivo

A genetically encoded calcium indicator for chronic in vivo two-photon imaging

Neurons in the nervous system can change their functional properties over time. At present, there are no techniques that allow reliable monitoring of changes within identified neurons over repeated experimental sessions. We increased the signal strength of troponin C-based calcium biosensors in the low-calcium regime by mutagenesis and domain rearrangement within the troponin C calcium binding moiety to generate the indicator TN-XXL. Using in vivo two-photon ratiometric imaging, we show that TN-XXL exhibits enhanced fluorescence changes in neurons of flies and mice. TN-XXL could be used to obtain tuning curves of orientation-selective neurons in mouse visual cortex measured repeatedly over days and weeks. Thus, the genetically encoded calcium indicator TN-XXL allows repeated imaging of response properties from individual, identified neurons in vivo, which will be crucial for gaining new insights into cellular mechanisms of plasticity, regeneration and disease