Fig. 1.

ES cells lack circadian bioluminescence rhythms. (A) Tol2 transposon-based Bmal1:luc reporter vectors were cotransfected with Tol2 transposase expression vector to establish the Bmal1:luc-pT2A stably transfected mouse ES cells. The obtained Bmal1:luc-pT2A stably transfected ES colonies exhibited apparent bioluminescence (Luc). (B) Photomultiplier-tubes (PMT)-based photon-counting assays were performed using Bmal1:luc ES cells after forskolin stimulation. Bioluminescence showed no circadian oscillation. (C) Microscopic bioluminescence image analysis. Single ES cells were plated onto a feeder cell layer and growing colonies raised from single cells were marked to trace the Bmal1:luc-driven bioluminescence, using an LV200 microscopic bioluminescence image analyzer (Movie S1 and Materials and Methods).