X chromosome reactivation and regulation in cloned embryos

Somatic cell nuclear transfer embryos exhibit extensive epigenetic abnormalities, including aberrant methylation and abnormal imprinted gene expression. In this study, a thorough analysis of X chromosome inactivation (XCI) was performed in both preimplantation and postimplantation nuclear transfer embryos. Cloned blastocysts reactivated the inactive somatic X chromosome, possibly in a gradient fashion. Analysis of XCI by Xist RNA and Eed protein localization revealed heterogeneity within cloned embryos, with some cells successfully inactivating an X chromosome and others failing to do so. Additionally, a significant proportion of cells contained more than two X chromosomes, which correlated with an increased incidence of tetraploidy. Imprinted XCI, normally found in preimplantation embryos and extraembryonic tissues, was not observed in blastocysts or placentae from later stage clones, although fetuses recapitulated the Xce effect. We conclude that, although SCNT embryos can reactivate, count, and inactivate X chromosomes, they are not able to regulate XCI consistently. These results illustrate the heterogeneity of epigenetic changes found in cloned embryos.     

Nuclear transfer-mediated rescue of the nuclear genome of nonviable mouse cells frozen without cryoprotectant

Nuclear transfer (NT) provides an opportunity for clonal amplification of a nuclear genome of interest. Here, we report NT-mediated reprogramming with frozen mouse cells that were nonviable because they were frozen at -80 degrees C for up to 342 days without a cryoprotectant. We derived eight embryonic stem (ES) cell lines from cloned blastocysts by conventional NT procedure and five ntES (nuclear transfer embryonic stem) cell lines by a modified NT procedure in which a whole cell instead of a nucleus was injected into an enucleated oocyte. Chromosome analysis revealed that 12 of 13 ntES cell lines have normal karyotypes. On injection of ntES cells into tetraploid blastocysts to generate clonal mice that are nearly completely ntES-cell derived, live pups were obtained; four clonal mice survived until adulthood. On injection of ntES cells into diploid blastocysts, chimeric mice with a high somatic ES cell contribution were generated; germ-line transmission was obtained. Our findings indicate that chromosome stability and genomic integrity can be maintained in mouse somatic cells after freezing without cryoprotection and that NT and ES cell techniques can rescue the genome of these cells.     

The region 3' to Xist mediates X chromosome counting and H3 Lys-4 dimethylation within the Xist gene

A counting process senses the X chromosome/autosome ratio and ensures that X chromosome inactivation (XCI) initiates in the female (XX) but not in the male (XY) mouse embryo. Counting is regulated by the X-inactivation centre, which contains the Xist gene. Deleting 65 kb 3' to Xist in XO embryonic stem (ES) cells affects counting and results in inappropriate XCI upon differentiation. We show here that normal counting can be rescued in these deleted ES cells using cre/loxP re-insertion, and refine the location of elements controlling counting within a 20 kb bipartite domain. Furthermore, we show that the 65 kb deletion also leads to inappropriate XCI in XY differentiated ES cells, which excludes the involvement of sex-specific mechanisms in the initiation of XCI. At the chromatin level, we have found that the Xist gene corresponds to a peak of H3 Lys-4 dimethylation, which is dramatically and specifically affected by the deletion 3' to Xist. Our results raise the possibility that H3 Lys-4 dimethylation within Xist may be functionally implicated in the counting process.     

The dynamics of imprinted X inactivation during preimplantation development in mice

In the mouse, there are two forms of X chromosome inactivation (XCI), random XCI in the fetus and imprinted paternal XCI, which is limited to the extraembryonic tissues. While the mechanism of random XCI has been studied extensively using the in vitro XX ES cell differentiation system, imprinted XCI during early embryonic development has been less well characterized. Recent studies of early embryos have reported unexpected findings for the paternal X chromosome (Xp). Imprinted XCI may not be linked to meiotic silencing in the male germ line but rather to the imprinted status of the Xist gene. Furthermore, the Xp becomes inactivated in all cells of cleavage-stage embryos and then reactivated in the cells of the inner cell mass (ICM) that form the epiblast, where random XCI ensues.     

Cloned mice and embryonic stem cell establishment from adult somatic cell

Cloning methods are now well described and becoming routine. Yet the frequency at which cloned offspring are produced remains below 2% irrespective of nucleus donor species or cell type. Especially in the mouse, few laboratories can make clones from adult somatic cells, and most mouse strains never succeed to produce cloned mice. On the other hand, nuclear transfer can be used to generate embryonic stem (ntES) cell lines from a patient's own somatic cells. We have shown that ntES cells can be generated relatively easily from a variety of mouse genotypes and cell types of both sexes, even though it may be more difficult to generate clones directly. Several reports have already demonstrated that ntES cells can be used in regenerative medicine in order to rescue immune deficient or infertile phenotypes. However, it is unclear whether ntES cells are identical to fertilized embryonic stem (ES) cells. In general, ntES cell techniques are expected to be applicable to regenerative medicine, however, these techniques can also be used for the preservation of the genetic resources of mouse strains instead of preserving such resources in embryos, oocytes or spermatozoa. This review seeks to describe the phenotype, application, and possible abnormalities of cloned mice and ntES cell lines.     

Progressive accumulation of epigenetic heterogeneity during human ES cell culture

Human embryonic stem cells (hESCs) can be maintained in culture over a large number of passages while maintaining apparently normal colony morphology. However, recent reports describe variability in epigenetic states in comparisons among different human ES cell lines. These epigenetic differences include changes in CpG methylation, expression of imprinted genes, and the status of X chromosome inactivation (XCI). We report here that the status of XCI in the female hESC line H9 (WA09) is hypervariable. We find that XIST expression can differ between individual culture isolates of H9. In addition, we find that XIST expression status can vary even between different colonies present within the same H9 culture, effectively rendering the culture mosaic. H9 cultures that lack XIST expression, but have cytological evidence of completed XCI, can also exhibit altered response to BMP4, a growth factor known to induce differentiation of hESCs to a trophectodermal lineage. In the same cultures we find biallelic expression of X-linked genes suggesting that these lines consist of mixtures of cells that retain inactivation of one of two X chromosomes following random choice. Prolonged culture of the XIST-negative isolates to high passage numbers did not result in changes in global epiproteomic signatures, demonstrating rather stable levels of post-translational nucleosome modifications within the culture-adapted hESC lines. The results show that epigenetic variants arise within human ES cell cultures after cell line derivation. In addition, the results indicate that apparently normal cultures of hESCs may contain mixtures of cells with differing epigenetic states. Assays of epigenetic integrity are warranted as quality control measures for the culture of hESCs.     

X inactivation counting and choice is a stochastic process: evidence for involvement of an X-linked activator

Female mammalian cells achieve dosage compensation of X-encoded genes by X chromosome inactivation (XCI). This process is thought to involve X chromosome counting and choice. To explore how this process is initiated, we analyzed XCI in tetraploid XXXX, XXXY, and XXYY embryonic stem cells and found that every X chromosome within a single nucleus has an independent probability to initiate XCI. This finding suggests a stochastic mechanism directing XCI counting and choice. The probability is directly proportional to the X chromosome:ploidy ratio, indicating the presence of an X-encoded activator of XCI, that itself is inactivated by the XCI process. Deletion of a region including Xist, Tsix, and Xite still results in XCI on the remaining wild-type X chromosome in female cells. This result supports a stochastic model in which each X chromosome in a nucleus initiates XCI independently and positions an X-encoded trans-acting XCI-activator outside the deleted region.     

The Probability to Initiate X Chromosome Inactivation Is Determined by the X to Autosomal Ratio and X Chromosome Specific Allelic Properties

Background

In female mammalian cells, random X chromosome inactivation (XCI) equalizes the dosage of X-encoded gene products to that in male cells. XCI is a stochastic process, in which each X chromosome has a probability to be inactivated. To obtain more insight in the factors setting up this probability, we studied the role of the X to autosome (X∶A) ratio in initiation of XCI, and have used the experimental data in a computer simulation model to study the cellular population dynamics of XCI.

Methodology/Principal Findings

To obtain more insight in the role of the X∶A ratio in initiation of XCI, we generated triploid mouse ES cells by fusion of haploid round spermatids with diploid female and male ES cells. These fusion experiments resulted in only XXY triploid ES cells. XYY and XXX ES lines were absent, suggesting cell death related either to insufficient X-chromosomal gene dosage (XYY) or to inheritance of an epigenetically modified X chromosome (XXX). Analysis of active (Xa) and inactive (Xi) X chromosomes in the obtained triploid XXY lines indicated that the initiation frequency of XCI is low, resulting in a mixed population of XaXiY and XaXaY cells, in which the XaXiY cells have a small proliferative advantage. This result, and findings on XCI in diploid and tetraploid ES cell lines with different X∶A ratios, provides evidence that the X∶A ratio determines the probability for a given X chromosome to be inactivated. Furthermore, we found that the kinetics of the XCI process can be simulated using a probability for an X chromosome to be inactivated that is proportional to the X∶A ratio. These simulation studies re-emphasize our hypothesis that the probability is a function of the concentration of an X-encoded activator of XCI, and of X chromosome specific allelic properties determining the threshold for this activator.

Conclusions

The present findings reveal that the probability for an X chromosome to be inactivated is proportional to the X∶A ratio. This finding supports the presence of an X-encoded activator of the XCI process.     

Improvement of mouse cloning using nuclear transfer-derived embryonic stem cells and/or histone deacetylase inhibitor

Nuclear transfer-derived ES (ntES) cell lines can be established from somatic cell nuclei with a relatively high success rate. Although ntES cells have been shown to be equivalent to ES cells, there are ethical objections concerning human cells, such as the use of fresh oocyte donation from young healthy woman. In contrast, the use of induced pluripotent stem (iPS) cells for cloning poses few ethical problems and is a relatively easy technique compared with nuclear transfer. Therefore, although there are several reports proposing the use of ntES cells as a model of regenerative medicine, the use of these cells in preliminary medical research is waning. However, in theory, 5 to 10 donor cells can establish one ntES cell line and, once established, these cells will propagate indefinitely. These cells can be used to generate cloned animals from ntES cell lines using a second round of NT. Even in infertile and "unclonable" strains of mice, we can generate offspring from somatic cells by combining cloning with ntES technology. Moreover, cloned offspring can be generated potentially even from the nuclei of dead bodies or freeze-dried cells via ntES cells, such as from an extinct frozen animal. Currently, only the ntES technology is available for this purpose, because all other techniques, including iPS cell derivation, require significant numbers of living donor cells. This review describes how to improve the efficiency of cloning, the establishment of clone-derived embryonic stem cells and further applications.