Analysis of Sex-specific imprint pattern and sex chromosome-linked gene activation. (A) Immunofluorescence of p57KIP2 in large cells derived from the OG2-SSC-Oct4/GFP cells (arrows). (B) Bisulfite genomic sequencing analyses of the DMR methylation status of parental origin-specific DNA methylation genes including Snrpn-DMR, Igf2-H19-DMR, and Dlk1-Meg3/Gtl2-DMR; filled ovals indicate methylated CpGs, and open ovals indicate unmethylatedCpGs (SSCs carry hypermethylation of Igf2-H19-DMR and Dlk1-Meg3/Gtl2-DMR but hypomethylation of Snrpn-DMR; this pattern was reversed in the OG2-SSC-Oocs). (C) Examination of the expression of sex chromosome-linked genes in OG2-SSC-Oocs: RT-PCR analyses showing that 7 testis-specific X-linked genes were significantly down-regulated (Magea, Fthi17, and Pramel3) or turned off (Usp26, Tex11, Tex13, and Tex16), three Y-linked genes (Usp9y, Ube1y, and Rbmy) were not expressed; GAPDH was used as the RT-PCR control. (D)RT-PCR analyses showing that oocyte-specific genes including X-linked genes (Usp9x and Bmp15) and autosomal gene GDF9 were turned on in OG2-SSC-Oocs; GAPDH was used as the RT-PCR control.