Fig. 1: A culture regimen and reporter cell line for naïve pluripotency in rabbit.

a Experimental scheme of B19_VAL, B19_VAL_14d, NaiveRep_KF, and NaiveRep_VAL cell line generation. b Immunostaining for SOX2, OCT4, DPPA5, OOEP, 5mC, H3K9me3, H3K14ac, and H3K4me3 in B19 cells before (_KF) and after (_VAL) switching to VALGöX culture medium. Scale bars: 50 µm for immunostainings, 100 µm for phase contrast. Violin plots show fluorescence intensity distribution. Means and standard deviations were calculated from measurements in at least 1,000 cells per condition (exact n indicated for each, from independent experiments: 3 for OCT4, OOEP, DPPA5, and 5mC; 4 for SOX2; 5 for H3K14ac and H3K4me3; 6 for H3K9me3). Comparisons between KF and VAL conditions were made using a two-sided Welch’s t-test for unequal variances. No significant differences were observed for SOX2 and H3K4me3 (difference between means <10%) and OCT4 (p = 0.35). Source data are provided in the Source Data file. c Heatmap of differentially expressed genes between B19_KF and B19_VAL cells (three biological replicates). d Phase-contrast images of B19 cells 48 h (_VAL) and 14 days (_VAL_14d) after transfer to VALGöX culture medium (5 experiments). Scale bars: 200 µm. e Heatmap of differentially expressed genes between B19_VAL and B19_VAL_14d cells. f Phase-contrast and epifluorescence images of NaiveRep_KF and NaiveRep_VAL cells (5 experiments). Scale bars: 100 µm. g Flow cytometry analysis of parental B19_KF, NaiveRep_KF, and NaiveRep_VAL showing blue and red fluorescence corresponding to EOS-tagBFP and DDPA2-mKO2, respectively. Basal tagBFP expression in NaiveRep_KF cells reflects endogenous ETn promoter activity downstream of the naïve-state-specific distal enhancer of OCT4/POU5F1 in the EOS vector²⁶.