Transcription factor Elf5, a key regulator of trophoblast lineage commitment, was up-regulated as early as day 1. A later marker of trophoblast differentiation, Tpbpa was detected at day 6. The expression levels of the pluripotency marker Oct4 gradually decreased over the course of the experiment. Trophoblast-directed differentiation of F121 knock down and control cell lines resulted in extensive cell death by day 3 after the induction for reasons unknown, thus precluding the analysis of mH2A function in the context of imprinted XCI. The preferential deposition of mH2A1 into the control regions of the inactive allele has been demonstrated for a subset of imprinted genes. The F1 hybrid genetic background of the F121 ESC line allowed for allelic discrimination based on SNP analyses, and we detected informative SNPs in expressed regions of Peg3 and Dlk1. The presence of the expressed SNPs was confirmed by the direct sequencing of the PCR products using genomic DNA as a template. RT-PCR results indicate that the Peg3 locus is not imprinted in undifferentiated ESCs. Bi-allelic expression was observed in all of the analyzed ESC lines, as both nucleotides could be detected at the SNP position in chromatogram traces. In contrast, Dlk1 demonstrated a skewed allelic expression status, with only a minor contribution from the imprinted allele. As expected, Dlk1 showed predominantly paternal expression, as the M.castaneus allele of paternal origin was the dominant one on the sequence traces. No difference in expression could be detected in knock down versus control ESC lines. The scientific literature contains a number of prominent studies that show that mH2As associate with the Xi in both mice and humans. However, both mice and humans harbor two genes encoding distinct mH2A histone variants. In addition, alternative splicing occurs for H2afy mRNAs in mice. Redundancy has complicated the production of knock out mice that lack all macroH2As. The targeted single gene deletion of mH2A1 in mice results in a surprisingly mild Metabolic Enzyme/Protease inhibitor phenotype, with animals that are viable and fertile, showing only subtle defects in glucose metabolism and lipid homeostasis. The fertility of male knock out mice Abmole BioScience PD 0332991 suggests that the inactivation of sex chromosomes in XY bodies of developing sperm is not dependent on mH2A1 alone, even though localization of this histone variant to XY bodies has been reported. Since mH2A1 knock out female mice are viable, XCI can clearly proceed in the absence of mH2A1 in vivo. In contrast, knock down of an mH2A2-like ortholog in zebrafish embryos resulted in severe and specific developmental defects.