三瀬 名丹

The tumor suppressor gene pten encodes a lipid phosphatase that dephosphorylates D3 of phosphatidylinositol(3,4,5)trisphosphate, producing phosphatidylinositol(4,5)bisphosphate. Although PTEN has been implicated in cell adhesion and migration, the underlying molecular mechanism is unknown. To investigate the role of PTEN in cell adhesion, we designed three different siRNAs (siRNA PTEN-a, siRNA PTEN-b, and siRNA PTEN-c) and transfected into 293T cells. Two days later, only the cells transfected with siRNA PTEN-b became round and detached from the culture dishes, whereas cells transfected with a control siRNA against GFP or the two other siRNAs against PTEN did not. Evaluation of the RNAi effect revealed that siRNA PTEN-b inhibited >95% of PTEN expression, the most effective among the three siRNAs. To check for non-specific effects such as interferon response and inhibition of off-target genes, we then used quantitative PCR analysis and DNA microarray analysis. None was detected, indicating that the RNAi system was highly specific. Immunofluorescence studies using PTEN-knockdown HeLa cells revealed that the loss of adhesion was accompanied by a reduction in the number of focal adhesion plaques and disorganization of the actin cytoskeleton. Transient and near-complete loss of PTEN expression induces loss of adhesion of the cells. (C) 2005 Elsevier Inc. All rights reserved.

That mammals can be cloned by nuclear transfer indicates that it is possible to reprogram the somatic cell genome to support full development. However, the developmental plasticity of germ cells is difficult to assess because genomic imprinting, which is essential for normal fetal development, is being reset at this stage. The anomalous influence of imprinting is corroborated by the poor development of mouse clones produced from primordial germ cells (PGCs) during imprinting erasure at embryonic day 11.5 or later. However, this can also be interpreted to mean that, unlike somatic cells, the genome of differentiated germ cells cannot be fully reprogrammed. We used younger PGCs (day 10.5) and eventually obtained four full-term fetuses. DNA methylation analyses showed that only embryos exhibiting normal imprinting developed to term. Thus, germ cell differentiation is not an insurmountable barrier to cloning, and imprinting status is more important than the origin of the nucleus donor cell per se as a determinant of developmental plasticity following nuclear transfer. (C) 2005 Wiley-Liss, Inc.

Prior to X-inactivation, Xist is transcribed in unstable form. The initiation of X-inactivation is associated with the appearance of stable Xist transcripts which coat the X chromosome to be inactivated. Using strand specific RT-PCR analysis of the 5' region of Xist, we have detected antisense transcripts (Xist AS) in undifferentiated embryonic stem (ES) cells, but not in female somatic cells. Screening of a female ES cell cDNA library allowed us to isolate one poly(A)-tailed cDNA clone corresponding to this RNA. 5' RACE analysis showed that XistAS and the P1 sense product of Xist overlap by at least 707 bp. Expression of XistAS was also detected in early mouse embryos before random X-inactivation in the epiblast lineage. Although XistAS is low in abundance, it may be involved in destabilizing Xist mRNA in undifferentiated ES cells. (C) 1999 Academic Press.

To clarify the sequence of events that ultimately achieves the nonrandom inactivation of the paternally inherited X chromosome in postpartum female mice heterozygous for T(X;16)16H, we set out to examine the expression of <i>Xist </i>alleles and the X-linked <i>HMG-lacZ</i> transgene in embryos recovered at the egg cylinder stage. Lack of expression of the <i>Xist<sup>b</sup></i> allele on the 16<sup>X</sup> translocation chromosome in the embryonic region of 7.5 d postcoitum (dpc) X<sup>16</sup>/X<sup>n</sup><i>Xist<sup>a</sup></i>;16<sup>X</sup><i>Xist<sup>b</sup></i>/16 embryos strongly suggested the occurrence of nonrandom inactivation in favor of the normal X chromosome. The simplest explanation would be biased choice, followed by postinactivation selection against genetically unbalanced cells. However, the frequency and distribution of β-galactosidase-positive cells in X<sup>16</sup>/X<sup>n</sup><i>lacZ</i>;16<sup>X</sup>/16 embryos at 6.5 and 7.5 dpc, together with earlier cytogenetic data, raised an intriguing possibility that the majority of 16<sup>X</sup> chromosomes were prevented from completing the inactivation process, when they had been chosen to be silenced. Phenotypes of female mice carrying a spontaneous recombination between X<sup>n</sup> and 16<sup>X</sup> in the segment defined by the T16H breakpoint and the X-linked <i>Ta </i>locus suggested that the nonrandomness was brought about by disruption of an X-chromosomal sequence or structure at the translocation breakpoint.

To clarify the sequence of events that ultimately achieves the nonrandom inactivation of the paternally inherited X chromosome in postpartum female mice heterozygous for T(X;16)16H, we set out to examine the expression of Xist alleles and the X-linked HMG-lacZ transgene in embryos recovered at the egg cylinder stage. Lack of expression of the Xist(b) allele on the 16(x) translocation chromosome in the embryonic region of 7.5 d postcoitum (dpc) X(16)/X(n)Xist(a);16(x)Xist(b)/ 16 embryos strongly suggested the occurrence of nonrandom inactivation in favor of the normal X chromosome, The simplest explanation would be biased choice, followed by postinactivation selection against genetically unbalanced cells. However, the frequency and distribution of beta-galactosidase-positive cells in X(16)/X(n)lacZ; 16(x)/16 embryos at 6.5 and 7.5 dpc, together with earlier cytogenetic data, raised an intriguing possibility that :he majority of 16(x) chromosomes were prevented from completing the inactivation process, when they had been chosen to be silenced. Phenotypes of female mice carrying a spontaneous recombination between X(n) and 16(x) in the segment defined by the T16H breakpoint and the X-linked Ta locus suggested that the nonrandomness was brought about by disruption of an X-chromosomal sequence or structure at the translocation breakpoint.

Mouse embryonal carcinoma (EC) cell lines are divided into two classes with or without the capability of reactivating the inactive X chromosome from a fusion partner of female lymphocyte. The 5' region of Xist was partially methylated in reactivating-competent EC cells but was fully methylated in reactivating-incompetent EC cells having a single X chromosome. Partial or heterogeneous methylation implies methylation of each CpG site in about half of the cell independently of methylation status of neighboring CpG sites. Fusion of the reactivating-competent EC cells with female lymphocytes induced not only de novo methylation in the 5' region of Xist allele on the hitherto inactivated X chromosome, but also demethylation of the same region of Xist on the other X chromosome from the female somatic cell. In contrast, no such changes occurred in hybrid cells involving reactivating-incompetent EC cells. Thus, partial methylation of the 5' region of Xist most probably maintained by low maintenance and high de novo methylation efficiency is correlated with reactivation potential of the EC cell. It is possible that this unique methylation pattern is implicated in random X inactivation in EC-hybrid cells in vitro and in epiblast cells in vivo. (C) 1996 Academic Press, Inc.

Xenopus laevis embryos which had been UV-irradiated as full-grown oocytes (UV-O) or after fertilization (UV-F) showed typical UV syndrome, namely dorsal axial deficiency. Morphological comparison revealed that UV-O embryos showed a clear dorsoventral polarity from early cleavage to gastrula stage, but UV-F embryos showed radially symmetrical development throughout embryogenesis. Although UV-O embryos developed morphologically normal-looking dorsal lips of the blastopore, they failed to develop dorsal axial structures at later stages. Implantation of dorsal lips demonstrated that the dorsal lip of UV-O embryos had less activity as Spemann's organizer than the dorsal lip of normal embryos. It is thus suggested that a morphological differentiation of the dorsal lip of the blastopore does not necessarily imply a functional differentiation of Spemann's organizer. Dorsal or ventral cytoplasm from normal embryos at the 8-16 cell stage was injected into a blastomere of UV-F and UV-O embryos at the same stage as the donor. The injection of the dorsal cytoplasm could rescue partially the UV syndrome of UV-F but not of UV-O embryos.

A new imprinted gene has been discovered in mice using the technique of restriction landmark genomic scanning (RGLS) with methylation sensitive enzymes. Eight out of 3,100 strain-specific NotI and BssHII spots were identified as imprinted in reciprocal F1 hybrids. Subsequently, we isolated a genomic clone for one locus on proximal chromosome 11 near the GIns locus, an imprinted region in uniparental disomic mice, and its corresponding cDNA clone. Expression of this transcript from the paternal allele was established using RT-PCR of reciprocal F1-hybrid mice. The amino-acid sequence deduced from the cDNA showed significant homology to the U2 small nuclear ribonucleoprotein auxiliary factor 35 kDa subunit.