Circadian Rhythms Monitoring via CRE-Luc2P Assay

A luciferase reporter driven by a tandem repeat of the Gpr19 CRE sequence (3 × CRE-Luc2P) was inserted between the ITR sequences of pAAV-MCS vector (Cell Biolabs Inc) to obtain pAAV-3 × CRE-Luc2P. HEK293T cells cultured in dish were co-transfected with pAAV-3 × CRE-Luc2P, pAAV-DJ, and pHelper according to the manufacturer's instructions (Cell Biolabs Inc). Three days after transfection, cells were harvested and resuspended in 1 ml of DMEM, followed by four freeze–thaw cycles and centrifugation. The titers of 3 × CREwt-Luc2P and 3 × CREmut-Luc2P virus solutions were ~ 8 × 10¹² genome copies/mL. The SCN slices were prepared according to our standard method^{59 (link)}. Two days after the preparation of SCN slices, the AAV solution (3 μL per slice) was inoculated on the surface of the SCN slices. Infected slices were further cultured for ~ 14 days. Thereafter, luminescence from the culture was measured with a dish-type luminometer (Kronos Dio, ATTO) at 35 °C using 1 mM luciferin^{59 (link)}. The luminescence was monitored for 2 min at 20-min intervals for each slice. The raw data were smoothed using a 1-h moving average and further detrended by subtracting a 24 h running average.