Our previous studies using restriction landmark genomic scanning (RLGS) defined tissue-or cellspecific DNA methylation profiles. It remains to be determined whether the DNA sequence compositions in the genomic contexts of the Not I loci tested by RLGS influence their tendency to change with differentiation. We carried out 3834 methylation measurements consisting of 213 Not I loci in the mouse genome in 18 different tissues and cell types, using quantitative real-time PCR based on a V IRTUAL IMAGE RLGS database. Loci were categorized as CpG islands or other, and as unique or repetitive sequences, each category being associated with a variety of methylation categories. Strikingly, the tissue-dependently and differentially methylated regions (T-DMRs) were disproportionately distributed in the non-CpG island loci. These loci were located not only in 5 ′ ′ ′ ′ -upstream regions of genes but also in intronic and non-genic regions. Hierarchical clustering of the methylation profiles could be used to define developmental similarity and cellular phenotypes. The results show that distinctive tissue-and cell type-specific methylation profiles by RLGS occur mostly at Not I sites located at non-CpG island sequences, which delineate developmental similarity of different cell types. The finding indicates the power of Not I methylation profiles in evaluating the relatedness of different cell types.

The isolation of stable trophoblast stem (TS) cell lines from early mouse embryos has provided a useful cell culture model to study trophoblast development. TS cells are derived from pre‐implantation blastocysts or from the extraembryonic ectoderm of early post‐implantation embryos. The derivation and maintenance of mouse TS cells is dependent upon continuous fibroblast growth factor (FGF) signaling. Gene expression analysis, differentiation in culture, and chimera formation show that TS cells accurately model the mouse trophoblast lineage. This unit describes how to derive, maintain, and manipulate TS cells, including DNA transfection and chimera formation. © 2015 by John Wiley & Sons, Inc.

The isolation of stable trophoblast stem (TS) cell lines from early mouse embryos has provided a useful cell culture model to study trophoblast development. TS cells are derived from pre‐implantation blastocysts or from the extraembryonic ectoderm of early post‐implantation embryos. The derivation and maintenance of mouse TS cells is dependent upon continuous fibroblast growth factor (FGF) signaling. Gene expression analysis, differentiation in culture, and chimera formation show that TS cells accurately model the mouse trophoblast lineage. This unit describes how to derive, maintain, and manipulate TS cells, including DNA transfection and chimera formation. Curr. Protoc. Stem Cell Biol. 7:1E.4.1‐1E.4.27. © 2008 by John Wiley & Sons, Inc.

The role of perchloric acid-soluble protein (PSP) was investigated in chick embryos. Fluorescently labeled anti-chick liver (CL)-PSP IgG was injected into the yolk sac in ovo at embryonic day 3, and became localized in neuroepithelial cells. Within 12 h, morphological changes were observed in 37.5% of anti-CL-PSP IgG-injected embryos, and the neuroepithelial cells formed a wavy line. No significant changes were observed in embryos injected with non-immune IgG or PBS. Increased expression of PCNA and decreased expression of neuronal class III b-tubulin were observed in the spinal cord after anti-CL-PSP IgG injection. These results suggest that PSP controls the proliferation and differentiation of neuroepithelial cells in chick embryos.