Fig. 2: Generation of embryos with parental-allele-specific DMR deletions from ESCs derived by Ha-Ha-fusion system.

a Scheme of generating embryos with parental-allele-specific DMR deletions. Hypomethylated DMRs were deleted by CRISPR-Cas9 in RFP-labeled AG-haESCs and GFP-labeled PG-haESCs. Diploid fused cells were obtained by fusing RFP-labeled AG-haESCs with GFP-labeled PG-haESCs with HVJ virus. 2N double-positive cells were enriched by FACS. Reconstructed embryos were generated by tetraploid complementation of fused cells. b Colony morphology and fluorescence labeling of diploid fused ESCs (top) and E3.0 reconstructed embryos (bottom). Scale bars, 200 µm. BF, bright field. c E9.5 (top) and E10.5 (bottom) reconstructed embryos (WT-DKO) carrying paternal H19-DMR and IG-DMR deletions. WT-DKO embryos were generated from late passages (P30) fused ESCs. The passage number of fused ESCs is inherited from the passage number of PG-haESCs. Scale bars, 500 µm. d DNA methylation levels at Xist-DMR in AG-haESCs, PG-haESCs and fused ESCs. Data are presented as mean ± SD, analyzed by Student’s t-test, *P < 0.05, ****P < 0.0001, ns, not significant. e Top, diagram showing deletion of Xist-DMR. Bottom, DNA sequences of PCR products amplified from Xist-DMR’s deleted region in Xist-DMR deletion PG-haESCs. f E9.5 (left) and E10.5 (right) reconstructed embryos (Xist-DKO) carrying paternal DKO and maternal Xist-DMR deletion. Scale bars, 1 mm. g A comparison of the developmental (the number of embryos in the amniotic sac divided by the number of embryos transferred) and alive rates of reconstructed embryos in c and f. Alive status is judged by the heartbeat. Data were generated from Table 1.