title: Expression and methylation of imprinted genes during in vitro differentiation of mouse parthenogenetic and androgenetic embryonic stem cell lines.
abstract: Messenger RNA and methylation levels of four imprinted genes, H19, Igf2r, Igf-2 and Snrpn were examined by northern and Southern blotting in mouse parthenogenetic, androgenetic and normal or wild-type embryonic stem cell lines during their differentiation in vitro as embryoid bodies. In most instances, mRNA levels in parthenogenetic and androgenetic embryoid bodies differed from wild type as expected from previously determined patterns of monoallelic expression in midgestation embryos and at later stages of development. These findings implicate aberrant mRNA levels of these genes in the abnormal development of parthenogenetic and androgenetic embryos and chimeras. Whereas complete silence of one of the parental alleles has previously been observed in vivo, we detected some mRNA in the corresponding embryonic stem cell line. This 'leakage' phenomenon could be explained by partial erasure, bypass or override of imprints, or could represent the actual activity status at very early stages of development. The mRNA levels of H19, Igf2r and Igf-2 and the degree of methylation at specific associated sequences were correlated according to previous studies in embryos, and thereby are consistent with suggestions that the methylation might play a role in controlling transcription of these genes. Paternal-specific methylation of the H19 promoter region is absent in sperm, yet we observed its presence in undifferentiated androgenetic embryonic stem cells, or before the potential expression phase of this gene in embryoid bodies. As such methylation is likely to invoke a repressive effect, this finding raises the possibility that it is part of the imprinting mechanism of H19, taking the form of a secondary imprint or postfertilization epigenetic modification necessary for repression of the paternal allele.
authors.affiliation: Division of Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010-0269.
authors.forename: P
authors.initials: P
authors.lastname: Szabó
authors.forename: J R
authors.initials: JR
authors.lastname: Mann
mesh_terms: D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D005260:Female; D015870:Gene Expression / Q000502:physiology; D008297:Male; D008745:Methylation; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D010312:Parthenogenesis / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D013234:Stem Cells / Q000502:physiology*
chemical_list: D017931:DNA Primers; D012333:RNA, Messenger