We therefore used a battery of cell biology approaches to determine the status of the XCI in the F m1-m2 ESC line, which contains a stable knock down of both mH2A1 and mH2A2. F m1-m2 female ESCs were induced to differentiate for 10 days in the presence of atRA, a differentiation procedure that is known to cause XCI in wild type female ESCs culminating in the formation of MCBs over the Xist RNA-FISH clouds. We employed immunofluorescence protocols to detect the presence of GDC-0449 Xi-specific epigenetic modifications, combined with an assay for subnuclear sites devoid of LDK378 customer reviews interphase transcription as judged by the absence of RNA Polymerase II. As expected, an mH2A1 signal was virtually absent in nuclei from F m1-m2 samples. However, presumptive inactive X chromosomes were detected as perinuclear DAPI-dense regions that were also devoid of RNA Pol II staining. In contrast, prominent MCBs were detected over the RNA Pol II exclusion zones in nuclei from a wild type F control line. Both mH2A1/mH2A2-deficient m1-m2) and wild type ) lines exhibited H3K27me3 staining coincident with regions lacking an RNA Pol II signal, corresponding to locations of Xi in interphase nuclei. In addition, Xist RNA-FISH demonstrated the presence of a single Xi located at peripheries of nuclei in both knock down and non-specific control lines. Two hundred cells were counted for the latter two assays, and the number of cells exhibiting XCI was unperturbed by the knock down of mH2A1/mH2A2. The pluripotency of double knock down and control cell lines was tested in vitro by the formation of embryoid bodies. All cell lines readily formed EBs by random aggregation, and gene expression analyses confirmed the presence of markers for all three germ layers, ectoderm, mesoderm, and endoderm. Female ESCs were slightly less efficient in up-regulating the mesoderm marker Myh6, while in male J1 ESCs this marker was strongly expressed in day 21 EBs. As expected, F121 transgenic knock down ESCs showed strong up-regulation of Xist expression at this EB stage, while the Xist expression in male ESC lines was virtually undetectable. Robust knock down of mH2A1.2 and mH2A2 was maintained in day 21 EBs. The differentiation-induced up-regulation of mH2A1.1 was observed in day 21 EBs in male samples, except for the universal mH2A1/mH2A2 knock down line J m1-m2, as expected.