Fig. 5. URI is essential for pluripotency maintenance.

(A) Scheme of conditional knockout URI lox mice. (B) Scheme of mESCs derivation from URI knockout mice treated with adenoviruses expressing either Cre recombinase combined with an enhanced green fluorescence protein EGFP (AdV-Cre-EGFP) or EGFP alone (AdV-EGFP) as control and cultured in presence of 2i/LIF. (C) Western blot (WB) of mESCs treated either with AdV-EGFP or AdV-Cre-EGFP as reported in (B). (D) IF of mESCs for URI and the totipotent-like marker ZSCAN4 after treatment with either Ad-EGFP or AdV-Cre-EGFP as reported in (B). Dashed outlines denote totipotent-like cells. Scale bar, 20 μm. (E) Abundance of the ZSCAN4 population in mESCs treated with either AdV-EGFP or AdV-Cre-EGFP as reported in from (D). t test; *P < 0.05. (F) qRT-PCR of transposable elements in mESCs treated either with AdV-EGFP or AdV-Cre-EGFP. Multi-t test; *P < 0.05. (G) qRT-PCR of totipotent-like genes in mESCs treated either with AdV-EGFP or AdV-Cre-EGFP. Multi-t test; *P < 0.05 and **P < 0.01. (H) Plasmid construct for permanent labeling of mESCs with EGFP. (I) BF merged images for endogenous EGFP signal in mESCs stably electroporated with plasmid from (H). Scale bar, 50 μm. (J) DNA electrophoresis of genotyping PCR for postinfected mESCs at the time of microinjection from (I). (K) Scheme of microinjection and tracking of labeled mESCs in 8C embryos. (L) Representative phase-contrast images for microinjected embryos as described in (K) showing endogenous EGFP signal for Z-stacked projection or single Z-slides in different embryos. Dashed orange outline denotes ICM compartment; yellow arrowheads point integrated cells outside the ICM in the TE compartment. Scale bar, 100 μm. (M) Frequency of microinjected mESCs in different compartment location from (L). (N) IF of microinjected embryos at blastocyst stage for GFP and CDX2 marker. Yellow dashed outlines identify integrated cells in TE. Scale bar, 100 μm.