Fig. 1

Design of the developed Oct4 tracing system. a Indicated cassettes were knocked into Oct4 (Pou5f1) and Rosa26 loci, using the CRISPR/Cas9 system. b Pou5f1 and Rosa26 loci after targeting with the above constructs. While Pou5f1 is constitutively active, tTR blocks expression from CAG-promoter in Rosa26 locus. This repression can be reversed by doxycycline (Dox) addition. c After the addition of 4-hydroxytamoxifen (4-OHT) to the cells of interest, derived from O4S ESCs, tTR:STOP cassette is excised by the Ert2CreErt2 recombinase. Expression of the FlpO recombinase is allowed from this time point. d When Oct4 gene becomes active, FlpO-mediated deletion of the Ert2CreErt2:STOP sequence occurs, resulting in cells permanently labeled with tdTomato regardless of subsequent Oct4 activity