Improved dynamic monitoring of transcriptional activity during longitudinal analysis in the mouse brain

Abstract

Bioluminescence imaging has proven to be a highly sensitive technique for assessing in vitro transcriptional activity toward understanding gene regulation patterns; however, application of this technique is limited for brain research. In particular, the poor spatiotemporal resolution is a major hurdle for monitoring the dynamic changes of transcriptional activity in specific regions of the brain during longitudinal analysis of living animals. To overcome this limitation, in this study, we modified a lentivirus-based luciferase glucocorticoid receptor (GR) reporter by inserting destabilizing sequence genes, and then the reporter was stereotaxically injected in the mouse infralimbic prefrontal cortex (IL-PFC). Using this strategy, we could successfully pin-point and monitor the dynamic changes in GR activity in IL-PFC during normal stress adaptation. The modified reporter showed a 1.5-fold increase in temporal resolution for monitoring GR activity compared to the control, with respect to the intra-individual coefficients of variation. This novel in vivo method has broad applications, as it is readily adaptable to different types of transcription factor arrays as well spanning wide target regions of the brain to other organs and tissues.

Decoding the temporal nature of brain GR activity in the NFκB signal transition leading to depressive-like behavior

“…The temporal resolution of the NFκB-Luc reporter, associated with the long half-life of luciferase [ 17 ], hindered the accurate monitoring of NFκB activation. Thus, we developed an improved NFκB-Luc2CP reporter by replacing Luc with Luc2CP as in the previously reported GRE-Luc2CP [ 16 ], expecting high temporal resolution with increased intensity. The monitoring of NFκB activity with this improved reporter showed clear dermal NFκB activation at 2 h post-LPS; however, there was still no NFκB activation observed in the IL-PFC (Fig.…”

“…To demonstrate this, we performed dual monitoring of GR activity with the GRE-Luc2CP reporter (Fig. 2a ) [ 16 ]. Unlike the differences shown in NFκB activation (Fig.…”

“…2 ). [ 1 , 16 ]. In the monitoring of NFκB and GR activities, three bioluminescence experiments were performed to investigate the LPS effect, the LPS effect on the RU486, or adrenalectomy (Supplementary Fig.…”

A Comprehensive Exploration of Bioluminescence Systems, Mechanisms, and Advanced Assays for Versatile Applications

“…The combination of these elements is referred to as rapid response genes, exhibiting a heightened reactivity to stimuli. Under moderate signal intensity, the luc2P gene demonstrates a swifter response compared to luc2, whereas the luc2CP gene exhibits rapid responsiveness with the least signal intensity [ 56 ]. These destabilized reporters give a response faster than nondestabilized reporters.…”