Journal: Nature Communications

Article Title: Efficient generation of germline chimeras in a non-rodent species using rabbit induced pluripotent stem cells

doi: 10.1038/s41467-025-60314-2

Figure Lengend Snippet: 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 vector .

Article Snippet: Spreads were stained with 1x DAPI for 15 min, mounted using Vectashield®, and examined with a Nikon Eclipse TI epifluorescence microscope at 63× magnification using a DAPI filter (Ex 377/50, Em 447/60).

Techniques: Immunostaining, Fluorescence, Flow Cytometry, Expressing, Activity Assay, Plasmid Preparation

Journal: Nature Communications

Article Title: Efficient generation of germline chimeras in a non-rodent species using rabbit induced pluripotent stem cells

doi: 10.1038/s41467-025-60314-2

Figure Lengend Snippet: a , b Gel electrophoresis of PCR amplification products for GFP DNA sequences in genomic DNA extracted from peripheral blood cells ( a ) and buccal swabs ( b ) of six viable chimeric animals (one biological replicate). c Confocal microscopy images of tissue sections from chimeras #A2 and #A5 showing GFP + cells labeled with anti-GFP antibody in muscle (scale bar: 250 µm), lung, liver, ovary (50 µm), skin (25 µm), and tongue (500 µm). Images represent three technical replicates per tissue. d Confocal image of a primary ovarian follicle labeled with anti-GFP in a section from female chimera #A6 (scale bar: 75 µm; three technical replicates). e Gel electrophoresis of PCR amplification products for GFP DNA sequences from genomic DNA of E14 F1 embryos obtained after insemination of female chimeras #A2 and #A6 with wild-type sperm (three technical replicates). f Epifluorescence images of whole-mount E14 F1 embryos and a non-chimeric control (one biological replicate). g Identification of four transgene integration sites in KEPi#28 donor cells and F1 fetuses via ligation-mediated PCR. Asterisks (*) indicate specific PCR bands confirmed by sequencing in KEPi#28 and F1 fetuses #16 and #19. Bands observed in other fetuses were not further analyzed. “ns” denotes non-specific PCR products with unreadable sequences (two technical replicates). h PCR amplification of genomic DNA from F1 fetuses confirming the integration of the GFP transgene at a Chromosome 1 locus identified in KEPi#28 cells via ligation-mediated PCR. REF, rabbit embryonic fibroblasts (negative control). Two technical replicates were performed.

Article Snippet: Spreads were stained with 1x DAPI for 15 min, mounted using Vectashield®, and examined with a Nikon Eclipse TI epifluorescence microscope at 63× magnification using a DAPI filter (Ex 377/50, Em 447/60).

Techniques: Nucleic Acid Electrophoresis, Amplification, Confocal Microscopy, Labeling, Control, Ligation, Sequencing, Negative Control