Fig 1.
Generation of Fgf5-P2A-Venus BAC Tg mice.
(A) Construction of the Fgf5-P2A-Venus BAC Tg. The Fgf5 BAC clone (RP23-153I24) covering 72 kb upstream and 112 kb downstream of Fgf5 gene was used. Note that the PGK-gb2-neo cassette was removed from the BAC construct prior to generation of Tg mice. P2A: porcine teschovirus-1 self-cleaving peptide; ipac: ires (internal ribosome entry site)-puromycin resistant gene. (B, C) Venus expression in WT and Tg embryos at E6.5 (line #2 and #571). Ex: extraembryonic region; Em: embryonic region. Scale bar: 100 μm.
Fig 2.
Venus expression in Fgf5-P2A-Venus BAC Tg embryo at E6.5.
(A) Immunofluorescence analysis of the Tg embryo at E6.5 for Oct3/4 (red), Venus (anti-GFP, green), Gata4 (purple) and Nuclei (Hoechst33342, blue). Higher magnification of optical sections is shown in panels X and Y. Note that Oct3/4 and Venus were co-expressed in the epiblast of the Tg embryo, while Gata4 expression was specifically observed in the endoderm regions of the Tg embryo. Venus expression was also seen in visceral endodermal layer. Open and closed arrowheads indicate endodermal and epiblast cells, respectively. Ex: extraembryonic region; Em: embryonic region; Epi: epiblast; En: endoderm. Scale bar: 50 μm. All images were captured by a Leica TCS-SP8 confocal microscope using a 40x/1.25 oil objective lens. (B) Whole-mount fluorescence in situ hybridization of Fgf5 in the Tg embryo at E6.5. Open arrowheads indicate cytoplasmic localization of endogenous Fgf5 mRNA. Scale bar: 20 μm. Images were captured by a Leica TCS-SP8 confocal microscope using a 40x/1.25 oil objective lens.
Fig 3.
Venus expression in Fgf5-P2A-Venus BAC Tg embryo at E7.5.
(A) Immunofluorescence images of the Tg embryo at E7.5 for Oct3/4 (red), Venus (anti-GFP, green), T (purple) and Nuclei (Hoechst33342, blue). Higher magnification of optical sections is shown in panels X and Y. Note that Oct3/4 and Venus were co-expressed in the epiblast of the Tg embryo, while T expression was observed in the mesodermal layer of the Tg embryo. Venus expression was also seen in visceral endodermal layer. Open and closed arrowheads indicate mesodermal and epiblast cells, respectively. Ex: extraembryonic region; Em: embryonic region; Epi: epiblast; Me: mesoderm; T: Brachyury/T. Scale bar: 50 μm. All images were captured by a Leica TCS-SP8 confocal microscope using a 20x/0.7 dry objective lens (projection images) and 40x/1.25 oil objective lens (section, X and Y images). (B) Whole-mount fluorescence in situ hybridization of Fgf5 in the Tg embryo at E7.5. Open arrowheads indicate cytoplasmic localization of endogenous Fgf5 mRNA. Scale bar: 50 μm.
Fig 4.
Derivation and characterization of Fgf5-P2A-Venus BAC Tg mEpiSCs.
(A) Immunofluorescence analysis of the Tg mEpiSCs for Venus (anti-GFP, green), Oct3/4 (purple), Nanog (red) and Nuclei (Hoechst33342, blue). Scale bar: 20 μm. All images were captured by a Leica TCS-SP8 confocal microscope using a 63x/1.4 oil objective lens. (B) RT-qPCR analysis of genes associated with pluripotency and lineage commitment in the Tg mEpiSCs and mESCs. β-actin was used as endogenous control for normalization. The mean and SD of three independent experiments are shown. *P < 0.05. (C) Venus expression in control and the Tg mEpiSCs was analyzed by flow cytometry.
Fig 5.
Reprogramming of Fgf5-P2A-Venus BAC Tg mEpiSCs into miPSCs.
(A) Experimental scheme for reprogramming of the Tg mEpiSCs into miPSCs. The Tg mEpiSCs stably expressing Klf5 or Nanog were cultured in NDiff227 medium supplemented with the Mek inhibitor (PD0325901), Gsk3 inhibitor (CHIR99021) and LIF. After 7 days, miPSC colonies were subjected to immunostaining. (B) Immunostaining for Venus (anti-GFP, green), CD31 (purple) and Nanog (red) in untransfected, vector control and miPSCs. OE: overexpression. Scale bar: 100 μm. (C) RT-qPCR analysis of Tg mEpiSCs and miPSCs. The mean and SD of two independent experiments are shown. *P < 0.05.
Fig 6.
Dynamic heterogeneity of Fgf5 expression in Fgf5-P2A-Venus BAC Tg mEpiSCs.
(A) Venus-positive and -negative populations were purified, cultured and subjected to flow cytometry analysis at indicated days. Note that Venus-positive and -negative cells were interchangeable within 48 h post culture, and Venus-positive and -negative cells could re-establish the original cell state within 6 and 4 days, respectively. (B) Gene expression was examined by RT-qPCR in Venus-positive and -negative cells. The mean and SD of three independent experiments are shown. *P < 0.05.