A Lentiviral Fluorescent Reporter to Study Circadian Rhythms in Single Cells

Abstract Circadian rhythms—self-sustained, ∼24-hour oscillations in transcript and protein levels—are generated by a cell-autonomous molecular clock. These rhythms shape how individual cells respond to external signals, influencing key decisions such as differentiation and apoptosis. However, current tools for visualizing circadian rhythms at the single-cell level often rely on genomic engineering and clonal expansion, limiting their accessibility and applicability. We present fluorescent circadian reporters based on the murine REVERBα/Nr1d1 gene, delivered via lentiviral transduction and compatible with time-lapse single cell microscopy. These reporters produce oscillatory signals that depend on a functional circadian clock and can be used to determine a cell’s circadian dynamics parameters, such as phase and period. Their simple and efficient delivery makes them suitable for a wide variety of cell types, greatly expanding opportunities to study single-cell circadian dynamics and their impact across diverse biological processes and systems.