Embryonal stem (ES) cell lines, established in culture from peri-implantation mouse blastocysts, can colonize both the somatic and germ-cell lineages of chimaeric mice following injection into host blastocysts. Recently, ES cells with multiple integrations of retroviral sequences have been used to introduce these sequences into the germ-line of chimaeric mice, demonstrating an alternative to the microinjection of fertilized eggs for the production of transgenic mice. However, the properties of ES cells raise a unique possibility: that of using the techniques of somatic cell genetics to select cells with genetic modifications such as recessive mutations, and of introducing these mutations into the mouse germ line. Here we report the realization of this possibility by the selection in vitro of variant ES cells deficient in hypoxanthine guanine phosphoribosyl transferase (HPRT; EC 2.4.2.8), their use to produce germline chimaeras resulting in female offspring heterozygous for HPRT-deficiency, and the generation of HPRT-deficient preimplantation embryos from these females. In human males, HPRT deficiency causes Lesch-Nyhan syndrome, which is characterized by mental retardation and self-mutilation.

Human preimplantation embryonic cells are similar in phenotype to cancer cells. Both types of cell undergo deprogramming to a proliferative stem cell state and become potentially immortal and invasive. To investigate the hypothesis that embryonic genes are re-expressed in cancer cells, we prepare ampli®ed cDNA from human individual preimplantation embryos and isolate embryospeci®c sequences. We show that three novel embryonic genes, and also the known gene, OCT4, are expressed in human tumours but not expressed in normal somatic tissues. Genes speci®c to this unique phase of the human life cycle and not expressed in somatic cells may have greater potential for targeting in cancer treatment. Oncogene (2001) 20, 8085 ± 8091.

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Adult intestinal epithelium consists of a sheet of single-cell thickness which is morphologically highly organized into tubular invaginations (crypts) and finger-like projections (villi). Proliferation of the cells is confined to the base of the crypts, from which cells migrate to the villi, where they are shed. The villi are formed during embryogenesis from a multilayered epithelium. In mice, crypts develop at about the time of birth from the epithelium between the villi, which by this stage is no longer multilayered. So far it has remained unknown how many progenitor cells contribute to each crypt, and whether they develop by the proliferation of already committed progenitors, or as a result of local inductive tissue interactions. Here, we have used mouse aggregation chimaeras as an experimental system to demonstrate immunohistochemically that the epithelium of individual crypts in small and large intestine of adult mice is always composed of cells of a single parental type. We have confirmed that this result is not an artefact of the chimaeric system by examining female mice that are mosaic for the X-linked alleles Pgk-1a and Pgk-1b. We conclude that the epithelium of each adult crypt is derived from a single progenitor cell.

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A HpaII-PCR assay was used to study DNA methylation in individual mouse embryos. It was found thatHpaII site H-7 in the CpG island of the X-chromosome-linked Pgk-1 gene is l10% methylated in oocytes and male embryos but becomes 40% methylated in female embryos at 6.5 days, about the time of X-chromosome inactivation of the inner cell mass.An assay based on the use of HpaII prior to the polymerase chain reaction (PCR) (20,22) allowed us to study DNA methylation in individual mouse embryos at the time of X-chromosome inactivation. We probed methylation in the CpG-rich island at the 5' end of the X-linked gene for phosphoglycerate kinase (Pgk-J), at a HpaII site (H-7) showing female-specific methylation in adult somatic tissues (Fig. 1). We found that site H-7 is not significantly methylated in male embryos but that in female embryos DNA methylation begins near 5.5 days and then rises, in both the embryo proper and the extraembryonic tissues, reaching nearly the adult level by 6.5 days.X-chromosome inactivation occurs in the inner cell mass of female mouse embryos between 5.5 and 6.5 days, at about the time of implantation (3, 10), a stage when the embryo consists of fewer than a thousand cells and the sex is not easily distinguishable. For this reason, only one prior study has been done correlating methylation with X-chromosome inactivation in the early embryo. Lock et al. (9) pooled many nonsexed embryos and found that methylation at sites in the first intron of the Hprt gene takes place between 9.5 and 13.5 days, well after the time of X-chromosome inactivation and the methylation we observe in the Pgk-1 gene.The promoter and first exon of Pgk-J are part of a CpG island that has been well characterized in both mice and humans (Fig. 1) (15) (Fig. 2). To assay for methylation, we made use of the fact that PCR amplification occurs only if the DNA between the two primer sites is intact. We chose PCR primers that flank H-7 and used a quantitative PCR procedure (20) to assay for HpaII-resistant (methylated) molecules. We had previously found, using this assay, that while male-embryo DNA was not amplified by PCR after HpaII digestion, female-embryo DNA was amplified, giving a signal 50% that of undigested DNA. Our interpretation was that site H-7 is methylated only on the inactive X chromosome (22). Figure 3 shows results of the HpaII-PCR assay on DNA from ectoplacental cone, extraembryonic ectoderm, and embryonic ectoderm dissected from two 6.5-day embryos, one male and one female. HpaII-resistant genomic molecules were present (giving band G in lanes marked +) in all three tissues of the female but not the male embryo. The larger fragments seen in the lanes without HpaII treatment came from M13 DNA, which was added as a carrier and also as a control for completeness of HpaII digestion. Band I in Fig. 3 is the PCR product of an internal standard, amplified by the same primers used for genomic DNA. The internal standard was a DNA fragment identical to genomic DNA except for a deletion internal to the primer binding sit...

Preferential paternal X-inactivation in the extra-embryonic tissues of the female mouse embryo is correlated with imprinted expression of the paternal allele of the Xist gene in pre-implantation development. Here we examine 11 CpG sites in Xist to determine whether differential methylation might be the molecular basis for imprinting. We find that three sites in the promoter region are methylated in eggs but not in sperm and that this differential methylation is maintained to the blastocyst stage when the paternal X-inactivation occurs. This is the first example of a primary gametic methylation imprint governing differential expression of parental alleles in pre-implantation embryos.

This review covers data on changing patterns of DNA methylation and the regulation of gene expression in mouse embryonic development. Global demethylation occurs from the eight-cell stage to the blastocyst stage in preimplantation embryos, and global de novo methylation begins at implantation. We have used X-chromosome inactivation in female embryos as a model system to study specific CpG sites in the X-linked Pgk-1 and G6pd housekeeping genes and in the imprinted regulatory Xist gene to elucidate the role of methylation in the initiation and maintenance of differential gene activity. Methylation of the X-linked housekeeping genes occurs very close in time to their inactivation, thus raising the question as to whether methylation could be causal to inactivation, as well as being involved in its maintenance. A methylation difference between sperm and eggs in the promoter region of the Xist gene, located at the X-chromosome inactivation centre, is correlated with imprinted preferential inactivation of the paternal X chromosome in extra-embryonic tissues. Based on our data, a picture of the inheritance of methylation imprints and speculation on the significance of the Xist imprint in development is presented. On a more general level, an hypothesis of evolution by "adaptive epigenetic/genetic inheritance" is considered. This proposes modification of germ line DNA in response to a change in environment and mutation at the site of modification (e.g., of methylated cytosine to thymine). Epigenetic inheritance could function to shift patterns of gene expression to buffer the evolving system against changes in environment. If the altered patterns of gene activity and inactivity persist, the modifications may become "fixed" as mutations; alternatively, previously silenced gene networks might be recruited into function, thus appearing as if they are "acquired characteristics." An extension of this hypothesis is "foreign gene acquisition and sorting" (selection or silencing of gene function according to use). "Kidnapping" and sorting of foreign genes in this way could explain the observation that increased complexity in evolution is associated with more "junk" DNA. Adaptive epigenetic/genetic inheritance challenges the "central dogma" that information is unidirectional from the DNA to protein and the idea that Darwinian random mutation and selection are the sole mechanisms of evolution.